Devices and methods for coupling mass spectrometry devices with chromatography systems

ABSTRACT

One embodiment of the invention provides a method of preparing an eluted sample containing salts or buffers from a liquid chromatography device for analysis by a mass spectrometry device. The method includes: continuously providing a non-polar solvent to the mass spectrometry device; receiving the eluted sample from the liquid chromatography device; flowing the eluted sample over a solid phase extraction column; flowing the non-polar solvent over the solid phase extraction column; and presenting non-polar solvent and the eluted sample to the mass spectrometry device.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/001,595, filed Nov. 2, 2007 and U.S. Provisional PatentApplication Ser. No. 61/001,597, filed Nov. 2, 2007. Each of thesepatent applications is hereby incorporated by reference in its entirety.

TECHNICAL FIELD

The present invention relates generally to high throughput screening offluidic samples, and more particularly, to automated systems and methodsfor increasing sample throughput of fluidic samples.

BACKGROUND ART

In many applications, such as drug discovery and development,environmental testing, and diagnostics, there is a need to analyze alarge number of samples in an efficient and reproducible manner. Many ofthe techniques used to analyze fluidic samples require that the samplesbe tested in a serial manner. In such applications, the process ofserial analysis can be automated through the use of a computercontrolled robotics and automation. Such devices are generally calledauto-injectors and are commonly interfaced to all manner of serialanalysis systems including, but not limited to, chromatography systems,mass spectrometers, and spectroscopic detectors.

Typical auto-injectors include a plurality of sample reservoirs, asyringe or syringe-like sample transport system, and an injection valvealong with the automation and computer control systems. Auto-injectorscommonly mimic cumbersome manual injection methods in which a meteredaliquot of a sample is aspirated from a desired sample reservoir into atransfer syringe. The aspiration process is often controlled by pullingback on a plunger or piston to create a negative pressure resulting inaspiration of the sample. The transfer syringe is then moved to anddocked with a stationary injection valve. The sample aliquot is thentransferred from the syringe to the injection valve by depressing thetransfer syringe plunger or activating the piston. The sample fills aninjection loop within the injection valve. Upon actuation of the valvethe sample is introduced into the fluidic circuit and diverted to theanalysis system.

The transfer syringe and the injection valve ports are then rinsed withan appropriate buffer or solvent to remove traces of the analyte tominimize contamination between samples. Contamination of the fluidicsystem with a sample can cause a significant barrier to the successfuloperation of a serial analysis system resulting in carryover andcompromised data. After an appropriate cleaning protocol the entireprocess is repeated for the next sample. Various embodiments of thisgeneral approach to auto-injectors are available commercially. Samplereservoirs used in auto-injectors range from glass vials to 96 or384-well microtiter plates. Sample reservoirs may be sealed with aplastic film or metal foil, or a septum. Some auto-injection devices useconventional syringes of various sizes attached to a robotic arm. Otherdevices use a tube attached to a small piston. The sample is aspiratedinto this tube and transferred to the injection valve. Some versions ofauto-injectors attempt to increase throughput by using multiple syringessuch that while an injection is being made by one syringe others arebeing washed. One auto-injector increases throughput with a simultaneousaspiration of eight samples. These samples are then loaded into thesample injection loops of eight separate injection valves. The samplesare then sequentially diverted from each of the eight injection valvesinto the analysis system. Throughput is thus increased through theparallelism of the process, however at increased cost and complexity.

Mass spectrometry (MS) with atmospheric pressure ionization (API) is acommonly used technique for the analysis of complex mixtures. Variationsof API-MS include electrospray ionization (ESI), atmospheric pressurechemical ionization (APCI) and atmospheric pressure photoionization(APPI). API-MS is used routinely in the pharmaceutical industry,environmental and forensic analysis, materials science, and inscientific applications. Both quantitative and qualitative informationabout specific compounds in complex mixtures can be obtained with theuse of API-MS methods.

However, API-MS has several drawbacks. Traditionally, MS is a serialprocess in which samples are analyzed sequentially unlike parallelanalysis schemes typically employed in many optical analysis systems.Sequential analysis can be impractical and in many cases economicallyunviable if very large numbers of samples are to be analyzed.

Furthermore, many compounds typically found at high concentrations incomplex biological, chemical, or environmental samples, such as salts,buffers, ionic or non-ionic detergents, proteins or enzymes, and othercofactors can cause a significant reduction in the amount of targetsignal observed in mass spectrometry. Interference from highconcentrations of non-volatile components are particularly troublesomebecause in addition to causing signal suppression non-volatile compoundstend to build up in the source region of the MS and gradually result ina decline in instrument performance.

The inherent expense involved in purchasing and operating massspectrometers makes it highly desirable to improve productivity bydevising methods and devices for increasing the analysis throughput(i.e. the number of samples that can be analyzed in a given time). Anymethod and device that attempts to increase throughput in API-MS mustaddress several key issues such as: (1) a rapid system for delivery of asample to the mass spectrometer must be designed; (2) the components ofcomplex mixtures that cause suppression of the target signal must beisolated and removed from the analytes of interest; (3) the non-volatilecomponents of complex mixtures that build up in the MS source and resultin a decay of instrument performance over time must be isolated andremoved; and (4) each sample must be cleaned from the analysis system toan acceptable level before the next sample is analyzed to preventsample-to-sample carryover that will result in contamination of thedata.

Liquid chromatography (LC) can be used to remove the salts, buffers, andother components from complex mixtures that may cause suppression of theMS signal of interest or result in degradation of MS instrumentperformance. Conventional liquid chromatography (LC) and its variations,such as high performance liquid chromatography (HPLC), typically involveflowing a liquid sample over a solid, insoluble matrix (generallyreferred to as Solid Phase Extraction (SPE)) commonly packed in a columnformat. The liquid sample includes an analyte(s) of interest that has anaffinity for the matrix under certain conditions of pH, saltconcentration, or solvent composition. Affinity of the analyte(s) ofinterest to the matrix may be due to hydrophobic or hydrophilicinteractions, ionic interactions, molecular size, or coordinationchemistry. In a highly specific variation, antibodies immobilized to thematrix are used to selectively capture molecules containing a highlyspecific epitope from complex mixtures.

As a result of the analyte(s) affinity to the matrix, the analyte(s)binds to the matrix and becomes immobilized while other (undesired)components of the liquid sample flow through the matrix and are removed.The analyte(s) of interest are then eluted away from the matrix bychanging the conditions of the flowing liquid, such that the analyte ofinterest no longer has affinity for the matrix. For example, changes inpH, ionic strength, solvent composition, temperature, and/or otherphysicochemical parameters may weaken the affinity of the analyte(s) forthe matrix.

However, the traditional use of liquid chromatography in high-throughputmass spectrometry has limitations. Very often, the throughput of aserial analysis is limited by the time it takes to collect the signalfrom an individual sample. In liquid chromatography applications, thematrix output signal from an analyte of interest is in the form of apeak, and the width of this peak in time is the ultimate determinant ofthe maximum throughput. A key factor in increasing mass spectrometrythroughput is the elution of the samples of interest from the insolublematrix as a tight, sharp band that is presented to the mass spectrometerin the shortest amount of time. For example, to achieve an overallthroughput greater than 30 seconds per sample, with baseline resolutionof each sample, the peak width must be narrower than 30 seconds. Asthroughput is increased, more stringent requirements on the peak widthmust be imposed. If the throughput begins to approach the peak width,the sequential samples begin to overlap, baseline resolution betweensamples in the MS is lost, and accurate quantification for each sampleis no longer possible.

In traditional LC, the analyte(s) of interest that are bound to theinsoluble solid matrix (typically packed in a column format) are elutedaway from the matrix by changing various properties of the liquidflowing over the matrix such that the analyte(s) are no longerimmobilized on the column. However, as the analyte(s) flow through thelength of the matrix a phenomenon known as band broadening occurs, inwhich linear diffusion causes the volume which contains the focusedanalyte(s) to expand. Consequently, the concentration of the analyte ofinterest presented to the mass spectrometer (or other analyzer) isdecreased, and a broad peak is produced that makes High ThroughputScreening (HTS) problematic.

SUMMARY OF THE INVENTION

The current invention describes a system and method for increasing thethroughput of analysis of selected components in complex biological,chemical, or environmental matrices with the use of, for example,chromatography and/or mass spectrometry. In various embodiments,throughput rates ranging from 30 seconds per sample to 1 second persample or faster are achievable, depending on the specific application.Further embodiments of the invention include an auto-injection systemthat increases throughput and minimizes sample carryover.

In accordance with one aspect of the invention, there is provided asystem for high throughput sample preparation and analysis. The systemincludes a chromatography column including an insoluble matrix. Afluidic circuit is capable of passing a fluid over the insoluble matrixin a first direction such that an analyte in the fluid binds to theinsoluble matrix, and back-eluting an elution fluid over the insolublematrix in a second direction opposite the first direction to output asample that includes the analyte. A controller controls the fluidiccircuit to periodically perform the steps of passing the fluid over theinsoluble matrix and back-eluting the elution fluid over the insolublematrix to output a plurality of samples at a periodic rate.

In accordance with related embodiments of the invention, the periodicrate is 30 seconds/sample or faster. The fluidic circuit may include avalving module capable of alternately directing fluid over the insolublematrix in the first direction and back-eluting an elution fluid over theinsoluble matrix in the second direction. The valving module may includeat least one pneumatically actuated valve and/or have an actuation timeof faster than 100 milliseconds.

In accordance with further related embodiments of the invention, thesystem may further include an analyzer for analyzing one or more of thesamples. The analyzer may be, for example, an optical analyzer or a massspectrometer that outputs a signal representative of the one or moresamples. The fluidic circuit may include a valve module that is actuatedto back-elute the elution fluid over the insoluble matrix, and whereinthe controller integrates the signal for a predetermined time after thevalve module is actuated to determine a characteristic of the sample.The mass spectrometer may include, without limitation, an electrosprayionization source, an atmospheric pressure chemical ionization source,or an atmospheric pressure photoionization source.

In accordance with still further related embodiments of the invention,the fluidic circuit may include tubing having a diameter between 20 μmto 300 μm. The fluidic circuit may include one or more surfaces whichcontact the fluid, wherein each surface is bioinert, such that it isnon-reactive. Each surface may include, for example, poly ether ketone,polyimide, titanium, and/or titanium alloy. The fluidic circuit mayinclude a fluidic pathway made of steel coated with a material tominimize binding with the analyte, such as polytetrafluoroethyleneand/or polyethylene glycol. The fluidic circuit may include an aspiratorfor aspirating an aliquot of the fluid to be passed over the insolublematrix. The chromatography column may include a first end and a secondend, wherein the analyte enters and exits the chromatography column atthe first end.

In accordance with another aspect of the invention, a method of highthroughput sample preparation and analysis includes passing a fluid overan insoluble matrix in a first direction, the fluid including an analytethat binds to the insoluble matrix. An elution fluid is back-eluted overthe insoluble matrix in a second direction opposite the first directionto output a sample that includes the analyte. The steps of passing thefluid and back-eluting the elution fluid are repeated so as to output aplurality of samples at a periodic rate.

In accordance with related embodiments of the invention, the periodicrate is 30 seconds/sample or faster. Analyzing each sample may includepresenting each sample to a mass spectrometer. Back-eluting may includesactuating a valving element to initiate flow of the elution fluid overthe insoluble matrix, wherein the method further includes integrating anoutput of the mass spectrometer for a predetermined time after the valveis actuated to determine a characteristic of the sample. Wash solutionmay be passed over the chromatography matrix prior to passing the fluidover the insoluble matrix, or back eluting the elution fluid. The fluidmay be aspirated from a fluid source prior to passing the fluid over aninsoluble matrix. The chromatography matrix may be packaged in a columnformat.

In accordance with still another aspect of the invention, a system forhigh throughput sample preparation and analysis includes a plurality ofchromatography columns and a mass spectrometer. A valve is capable ofselectively presenting effluent from one of the plurality ofchromatography columns to the mass spectrometer.

In accordance with related embodiments of the invention, the valve maybe actuated to present effluent from one of the plurality ofchromatography columns to the mass spectrometer. A processor may receivean output signal from the mass spectrometer, and integrate the outputfor a predetermined time after the valve is actuated to determine acharacteristic of the sample.

In still other aspects of the invention, a computer program product ispresented for use on a computer system for controlling a high throughputsystem having a fluidic circuit in fluid communication with achromatography column. The computer program product includes a computerusable medium having computer readable program code thereon. Thecomputer readable program code includes program code for controlling thefluidic circuit to pass a fluid over the insoluble matrix in a firstdirection such that an analyte in the fluid binds to the insolublematrix. The computer readable program code also includes program codefor controlling the fluidic circuit to back-elute an elution fluid overthe insoluble matrix in a second direction opposite the first directionto output a sample that includes the analyte; and program code forrepeating the passing of the fluid and the back-eluting the elutionfluid to output samples at a periodic rate.

In accordance with related embodiments of the invention, the computerreadable program code for controlling the fluidic circuit to back-elutethe elution fluid includes program code for actuating a valve modulethat allows the elution fluid to flow through the chromatography columnin the second direction. The high throughput system may include a massspectrometer for analyzing the sample, wherein the computer programproduct further includes program code for integrating an output of themass spectrometer upon actuation of the valve module to determine acharacteristic of the sample.

In accordance with another embodiment of the invention, anauto-injection system for high throughput screening of fluidic samplesincludes a sample sipper tube, a sample loop, and an injection valve.The injection valve applies a reduced pressure to the sample sippertube. When the injection valve is in a first position, the sample loopis in fluid communication with the sample sipper tube.

In related embodiments of the invention, the system may further includea vacuum means for supplying the reduced pressure. The vacuum means mayinclude a vacuum pump for continuous application of reduced pressureand/or a piston for metered application of reduced pressure. A valve mayselect one of the vacuum pump and the piston pump as a source of thereduced pressure. An inline trap may be positioned between the vacuummeans and the injection valve. A cutoff valve, which may be a solenoidvalve, may meter an amount of sample fluid to be aspirated into thesample loop via the sample sipper tube, the cutoff valve positionedbetween the vacuum means and the injection valve. Fluid contactingsurfaces of the system may be made of a material from the group ofmaterials consisting of polytetrafluoroethylene (available under theTEFLON® trademark from E. I. Du Pont De Nemours and Company ofWilmington, Del.), fused silica, and poly ether ketone.

In further related embodiments of the invention, when the injectionvalve is in a second position, the sample loop is in fluid communicationwith an output port of the injection valve. When the injection valve isin the second position, the sample sipper tube may be in fluidcommunication with a source of the reduced pressure so as to aspiratewash fluid, an inline-trap capturing the wash fluid.

In accordance with still another aspect of the invention, anauto-injection system for high throughput screening of fluidic samplesincludes a source of reduced pressure, a sample loop, a sample sippertube, and an injection valve. The injection valve includes a first portin fluid communication with the sample sipper tube; a second port influid communication with the sample loop; a third port in fluidcommunication with the sample loop; and a fourth port in fluidcommunication with the source of reduced pressure.

In related embodiments of the invention, when the injection valve is ina first position the source of reduced pressure, the sample loop, andthe sample sipper tube are in fluid communication. The injection valvemay include a fifth port for outputting sample fluid from the sampleloop. When the injection valve is in a second position, the sample loopis in fluid communication with the fifth port. The system may include asource of high pressure, and wherein the injection valve furtherincludes a sixth port in fluid communication with the source of highpressure.

The source of reduced pressure may include a vacuum pump and/or apiston. A valve may select one of the vacuum pump and the piston pump asa source of the reduced pressure. An inline trap may be positionedbetween the source of reduced pressure and the injection valve. When theinjection valve is in a second position, the sample sipper tube may bein fluid communication with the source of the reduced pressure so as toaspirate wash fluid, the inline-trap capturing the wash fluid. A cutoffvalve, such as a solenoid valve, may be used for metering an amount ofsample fluid to be aspirated into the sample loop via the sample sippertube, the cutoff valve positioned between the source of reduced pressureand the injection valve.

In accordance with another embodiment of the invention, an autosamplersystem for repetitive sampling and presentation of samples includes afluidic circuit. The fluidic circuit includes a sample port in fluidcommunication with an injection valve. The fluidic circuit furtherincludes means for applying a reduced pressure to the sample port toload a sample into the fluidic circuit. The sample is presented, viaoutput means, into an analyzer from an output port of the fluidiccircuit that is distinct from the sample port. The system furtherincludes automated means for positioning the multiple samples relativeto the sample port.

In related embodiments of the invention, the means for applying areduced pressure may include a trap, and/or may continuously apply anegative pressure to the sample port throughout the presentation ofsamples. The automated means for positioning multiple samples mayinclude a robotic device for successively presenting wells ofmicroplates. The samples are processed at a rate of greater than onesample every 30 seconds. The analyzer may be a mass spectrometer. Thesample may be aspirated intermittently into the sample port, while fluidis continuously injected into the analyzer.

In further related embodiments of the invention, the fluidic circuit mayinclude a resin for purification of the samples. The system may furtherinclude means for introduction of a sample to the resin, washing theresin with a wash solution and back-eluting the sample with an elutionsolution prior to presentation.

In accordance with another embodiment of the invention, a system forhigh throughput sample preparation and analysis includes achromatography column including an insoluble matrix. Fluidic circuitmeans passes a fluid over the insoluble matrix in a first direction suchthat an analyte in the fluid binds to the insoluble matrix, and passesan elution fluid over the insoluble matrix to output the analyte to ananalyzer. A controller controls the fluidic circuit to periodicallyperform the steps of passing the fluid over the insoluble matrix andpassing the elution fluid over the insoluble matrix to output to theanalyzer a plurality of samples at a periodic rate, such that thefluidic circuit presents only at least one of the elution fluid and theanalyte to the analyzer.

In accordance with another embodiment of the invention, a system forhigh throughput screening of fluid samples includes a sample aspirationtube, a valving element, sample loop, and an analyzer. A controllercontrols the valving element to alternatively aspirate a first fluidinto the sample loop via the sample aspiration tube, and aspirate asecond fluid via the aspiration tube while simultaneously outputting thefirst fluid in the sample loop to the analyzer.

In another embodiment auto-injection system provides high throughputscreening of fluidic samples. A sample injection valve has a firstposition which applies a reduced pressure to a sample sipper tube foraspirating a fluidic sample into the sample sipper tube, and a secondposition which delivers the fluidic sample to a sample supply loop. Acolumn control valve has a first position which delivers the fluidicsample from the sample supply loop to a sample chromatography column,and a second position which reverses direction of fluid flow through thesample chromatography column to deliver the fluidic sample to a sampleanalyzer, e.g., a mass spectrometer. A wash control valve has a firstposition which supplies a wash buffer solution to the samplechromatography column in a forward fluid flow direction, and a secondposition which supplies elution solvent to flush the sample supply loop.Positioning means present individual microplate sample wells to thesample sipper tube. Automated control means creates a cycle ofrepeatedly introducing samples and actuating the sample injection valve,column control valve, and wash control valve.

In a further such embodiment, the first position of the sample injectionvalve may further deliver wash buffer solution from the wash controlvalve to the sample chromatography column. The second position of thesample injection valve may further deliver elution solvent from the washcontrol valve to the sample supply loop. The first position of thecolumn control valve may further supply elution solvent to flush thesample analyzer. The second position of the column control valve mayfurther allow elution solvent to exit from the sample supply loop. Thesecond position of the wash control valve may further deliver washbuffer solution to flush the sample supply loop.

In a further embodiment, a wash buffer supply pump provides wash buffersolution to the wash control valve. A first elution solvent pump mayprovide elution solvent to the column control valve. And a secondelution solvent pump may provide elution solvent to the wash controlvalve.

Embodiments also include a similar method of performing high-throughputscreening of fluidic samples. A sample injection valve, a column controlvalve, and a wash control valve, each having two operating positions,are provided and arranged to perform a repeating cycle for highthroughput screening of fluidic samples. The cycle includes positioningeach of the valves in a first operating position in which: (i) a fluidicsample in a microplate sample well is presented to a sample sipper tubeand aspirated through the sample sipper tube to the sample injectionvalve, (ii) elution solvent is supplied by the column control valve to asample analyzer, and (iii) wash buffer solution is delivered from eachof the valves in series to a sample chromatography column forequilibration of the column. The sample injection valve is actuated to asecond operating position in which: (i) the sample sipper tube iswithdrawn from the fluidic sample and the aspirated sample is deliveredby the sample injection valve through a sample supply loop to thecolumn, and (ii) wash buffer solution is delivered from each of thevalves in series to the column for purification of the sample. Thecolumn control valve and the wash control valve are actuated torespective second operating positions in which: (i) the sample sippertube aspirates a wash solution for cleaning, (ii) elution solvent issupplied through each of the valves in series to flush the sample supplyloop, and (iii) elution solvent is supplied by the column control valveto reverse direction of fluid flow through the column to deliver thefluidic sample to the analyzer. Finally, each of the valves is actuatedback to their respective first operating positions to repeat the cycle.

In a further such embodiment, the cycle is performed at a speed ofgreater than two samples per minute. The sample analyzer may be a massspectrometer.

In another embodiment a sample injection system includes a vacuumsource, a conduit in communication with the vacuum source, a fluidsensor configured to detect the presence of the fluid in the conduit, asample loop in communication with the conduit; and a sipper incommunication with the sample loop.

The above embodiment can include several additional features. Theconduit can include a transparent portion. The fluid sensor can be anoptical sensor and configured to detect the presence of fluid in thetransparent portion of the conduit. The system can include a traplocated between the vacuum source and the conduit. A valve can becoupled to the sipper, the sample loop, and the conduit. The valve canbe a multi-port valve. The valve can be pneumatically, electrically,electromechanically, or mechanically actuated. The valve can beconfigured to interrupt fluid communication between the conduit and thesample loop when fluid is detected by the fluid sensor.

The system can include a robotic system for positioning the sipper toaspirate a fluid sample from a sample reservoir. The robotic system canbe configured to lower the sipper into the sample reservoir until fluidis detected by fluid detector. The robotic system can be furtherconfigured to prevent the sipper from traveling beyond a definedposition. The defined position can be specified by a user. The roboticsystem can be configured to retract the sipper from the reservoir whenfluid is detected by the fluid sensor.

Another embodiment is directed to an auto-injection system for highthroughput screening of fluidic samples. The system includes a vacuumsource, a sample injection valve, a conduit connecting the vacuum sourceand the sample injection valve, a fluid sensor configured to detect thepresence of the fluid in the conduit, a column control valve configuredto facilitate a continuous flow of an elution solvent to a sampleanalyzer, a wash control valve, and automated control means for creatinga cycle of repeatedly introducing samples and actuating the sampleinjection valve, column control valve, and wash control valve.

The sample injection valve has a first position which applies a reducedpressure to a sipper for aspirating a fluidic sample into the sipper,and a second position which delivers the fluidic sample from the samplesupply loop.

The column control valve has a first position which simultaneouslydelivers the fluidic sample from the sample supply loop to a samplechromatography column in a first direction and delivers an elutionsolvent to the sample analyzer, and a second position which flows theelution solvent over the sample chromatography column in a seconddirection to deliver the fluidic sample and the elution solvent to thesample analyzer.

The wash control valve has a first position which supplies a wash buffersolution to the sample chromatography column in a forward fluid flowdirection, and a second position which supplies elution solvent to flushthe sample supply loop.

The above embodiment can include several additional features. Theconduit can include a transparent portion. The fluid sensor can be anoptical sensor and configured to detect the presence of fluid in thetransparent portion of the conduit.

The system can include a robotic system for positioning the sipper toaspirate a fluid sample from a sample reservoir. The robotic system canbe configured to lower the sipper into the reservoir until fluid isdetected by fluid detector. The robotic system can be configured toprevent the sipper from traveling beyond a defined position. The definedposition can be specified by a user. The robotic system can beconfigured to retract the sipper from the reservoir when fluid isdetected by the fluid sensor.

Another embodiment is directed to a method of high-throughput sampleinjection comprising providing a vacuum source, a conduit incommunication with the vacuum source, a fluid sensor configured todetect the presence of fluid in the conduit, a sample loop incommunication with the conduit, and a sipper in communication with thesample loop; applying suction to the sipper; inserting the sipper into asample reservoir; and withdrawing the sipper from the sample reservoirupon detection of fluid by the fluid sensor.

The above embodiment can include several additional features. The methodcan include withdrawing the sipper from the sample reservoir uponadvancement beyond a defined position. The method can also includereporting an error. The method can further include analyzing a sampleheld in the sample loop.

Another embodiment of the invention provides a method of preparing aneluted sample containing salts or buffers from a liquid chromatographydevice for analysis by a mass spectrometry device. The method includes:

continuously providing a non-polar solvent to the mass spectrometrydevice; receiving the eluted sample from the liquid chromatographydevice; flowing the eluted sample over a solid phase extraction column;flowing the non-polar solvent over the solid phase extraction column;and presenting non-polar solvent and the eluted sample to the massspectrometry device.

This embodiment can have several variations. For example, the liquidchromatography device can be an ion exchange chromatography device. Theliquid chromatography device can be a cation exchange chromatographydevice. The liquid chromatography device can be a size exclusionchromatography device. The method can also include the step of opticallyanalyzing the eluted sample from the liquid chromatography device togenerate an optical data set. The method can also include the step ofassociating the optical data set with a data set generated by the massspectrometry device. The method can also include the step of flowing apolar wash solution over the solid phase extraction column.

Another embodiment of the invention provides a sample injection systemfor coupling a liquid chromatography device with a mass spectrometrydevice. The system can include a sample injection valve and a columncontrol valve. The sample injection valve can include (i) a firstposition that allows sample from the liquid chromatography device topass through the sample injection system, and (ii) a second positionthat loads sample from the liquid chromatography device onto a samplesupply loop. The column control valve can include (i) a first positionthat simultaneously delivers the fluidic sample from the sample supplyloop to a solid phase extraction column in a first direction anddelivers an elution solvent to the sample analyzer, and (ii) a secondposition that flows the elution solvent over the solid phase extractioncolumn in a second direction to deliver the fluidic sample and theelution solvent to the sample analyzer.

This embodiment can have several variations. The system can also includean optical detector for analyzing the sample from the liquidchromatography device. The system can also include a diversion valvelocated between the liquid chromatography device and the sampleinjection valve. The diversion valve can be actuated as a result ofsignal generated by the optical detector. The system can also include afraction collector. The elution solvent can be a polar solvent or anon-polar solvent.

Another embodiment of the invention provides a method of preparing aneluted sample from a liquid chromatography device for analysis by a massspectrometry device. The method includes: continuously providing a polarsolvent to the mass spectrometry device, receiving the eluted samplefrom the liquid chromatography device; flowing the eluted sample over aHILIC column; flowing the polar solvent over the HILIC column; andpresenting the polar solvent and the eluted sample to the massspectrometry device.

The liquid chromatography device can be one selected from the groupconsisting of: an ion exchange chromatography device, a cation exchangechromatography device, and a size exclusion chromatography device. Themethod can also include the step of optically analyzing the elutedsample from the liquid chromatography device to generate an optical dataset. The method can also include the step of associating the opticaldata set with a data set generated by the mass spectrometry device. Themethod can also include the step of flowing a polar wash solution overthe solid phase extraction column.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing features of the invention will be more readily understoodby reference to the following detailed description, taken with referenceto the accompanying drawings, in which:

FIG. 1 is a block diagram of a rapid chromatography system, inaccordance with an embodiment of the invention;

FIG. 2( a) is a schematic of a rapid chromatography system that includestwo injection valves, in accordance with an embodiment of the invention;

FIG. 2( b) is a schematic of the rapid chromatography system of FIG. 2(a) when a complex mixture is passed through a matrix in a firstdirection, in accordance with an embodiment of the invention;

FIG. 2( c) is a schematic of the rapid chromatography system of FIG. 2(a) when elution fluid is passed through the matrix in a seconddirection, in accordance with an embodiment of the invention; and

FIG. 3 is a schematic of a multiplexed analyzer system, in accordancewith an embodiment of the invention.

FIG. 4 is a schematic of an auto-injection device, in accordance with anembodiment of the invention.

FIG. 5( a) is a schematic of the auto-injection device of FIG. 4 duringsample aspiration, in accordance with an embodiment of the invention.

FIG. 5( b) is a schematic of the auto-injection device of FIG. 4 whenaspirated sample is output to a fluidic circuit, in accordance with anembodiment of the invention.

FIGS. 6(A)-6(D) is a schematic of an embodiment using three valves.

FIG. 6(E) depicts an embodiment of a three-valve auto-injective device.

FIG. 6(F) is an isometric projection of a bracket for supporting athree-valve auto-injection device.

FIG. 7 is a schematic of an auto-injection device incorporating a fluidsensor, in accordance with an embodiment of the invention.

FIG. 8( a) is a schematic of the auto-injection device of FIG. 7 beforesample aspiration, in accordance with an embodiment of the invention.

FIG. 8( b) is a schematic of the auto-injection device of FIG. 7 duringsample aspiration, in accordance with an embodiment of the invention.

FIG. 8( c) is a schematic of the auto-injection device of FIG. 7 whenaspirated sample is output to a fluidic circuit, in accordance with anembodiment of the invention.

FIG. 9 is a flow chart illustrating the operation of a high-throughputsample injection system, in accordance with an embodiment of theinvention.

FIGS. 10-11( e) are schematics of a system for coupling a massspectrometry system and a liquid chromatography system.

FIG. 12 is a schematic of a method for processing an eluted sample froma liquid chromatography system in a mass spectrometry device

DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

In illustrative embodiments, an automated system and method forincreasing sample throughput and/or analysis of selected components incomplex biological, chemical, or environmental matrices is presented.Generally, the system includes a chromatography column and fluidiccircuit that is capable of rapidly outputting a plurality of samples toan analyzer, such as a mass spectrometer. In various embodiments, samplethroughput rates ranging from 30 seconds per sample to 1 second persample or faster are achievable, depending on the specific application.Further embodiments of the invention include an auto-injection systemthat increases throughput and minimizes sample carryover. Details arediscussed below.

FIG. 1 shows a block diagram of a rapid chromatography system 100 forrapidly outputting an analyte of interest while removing undesirablesalts, buffers, and other components from a complex mixture, inaccordance with one embodiment of the invention. Such undesirablecomponents may, for example, degrade analyzer performance or cause anoutput signal from an analyzer to be suppressed.

The system 100 relies on the principle of back-elution for the specificpurpose of increasing sample throughput. In particular, the complexmixture to be analyzed is delivered to an insoluble matrix 102 in afirst direction 109 via a fluidic circuit 104. The matrix 102, which maybe packed in a chromatography column, is selected such that theanalyte(s) of interest is selectively immobilized. The matrix 102 maybe, without limitation, various resins known in the art ofchromatography. Typically, the analyte binds to the first part of thematrix 102 encountered due to a phenomenon known as focusing. Infocusing, a large amount of the analyte may be immobilized in a verysmall physical space within the head of the matrix 102 due to a strongaffinity for that matrix 102.

Non-binding components of the complex mixture, which may include,without limitation, salts, buffers, and/or detergents, are not soimmobilized and pass through the insoluble matrix 102. These undesirablecomponents are typically diverted to waste by the fluidic circuit 104.To ensure sufficient removal of the undesirable components, the matrix102 may be washed for a predetermined period of time, while theanalyte(s) of interest is still immobilized and focused on the head ofthe matrix.

Once the undesirable components have been removed from the matrix,elution fluid is passed via the fluidic circuit 104 over the matrix 102such that the analyte(s) is no longer immobilized by the matrix 102.However, instead of passing the elution fluid over the matrix 102 in thefirst direction 109, the elution fluid is passed over the matrix 102 ina second direction 111 that is substantially opposite the firstdirection 109, in accordance with preferred embodiments of theinvention. Thus, the analyte(s) does not travel through the length ofthe matrix 102, but is instead back-eluted from the matrix 102 in theopposite direction it was loaded. Due to the focusing effect, theanalyte(s) does not have to travel through the entire bed of thechromatography matrix 102, and a minimal amount of linear diffusiontakes place. Thus, a sharp, concentrated sample peak can be output fromthe matrix 102 within a minimal bandwidth of time. The sharp sample peakobtained by back-elution is significantly sharper than those obtainedwhen using conventional chromatography. The samples back-eluted from thematrix 102, which contain the analyte(s) of interest, can subsequentlybe introduced into an analyzer 116 in a rapid and concentrated manner.

In various embodiments of the invention, a controller 125 automaticallycontrols the fluidic circuit 104 to periodically perform the steps ofpassing the fluid over the matrix 102 in the first direction andback-eluting the elution fluid over the matrix 102 in the seconddirection, so as to obtain a high sample-throughput rate. The controller125 may include, without limitation, a processor which may beappropriately pre-programmed or configured to be loaded with anappropriate program. The controller 125 may work in conjunction with arobotic system that samples an aliquot of the complex mixture to beanalyzed, and that allows for sequential presentation of each complexmixture to be analyzed.

Various methodologies may be used in which containers of each complexmixture to be analyzed and a sipper tube can be moved relative to oneanother to allow for sequential sampling. These methodologies include,but are not limited to, systems in which the containers of the liquid tobe analyzed (e.g., a microtiter plate or an array of vials) are held ina fixed position and the sipper tube is translocated by means of arobotic arm to sequentially sample each container. Robotic microplatepositioning systems are known in the art as in U.S. Pat. No. 5,985,214.Preferably, the robotic system should be capable of presenting samplesat a rate that does not limit the throughput of the device. In otherembodiments, the sipper tube can be immobilized and each container to beanalyzed can be moved into a position where an aliquot can be sampled.In an embodiment, liquid samples can be transported to the sipper tubewith the use of a laminated tape or belt system for sequential analysis,as described in the following U.S. patents and patent applications: U.S.Patent Application Publication No. 2002/0001544, U.S. Patent ApplicationPublication No. 2003/0119193, and U.S. Pat. No. 6,812,030. Systems thatincorporate elements of both approaches (e.g., moving the samplecontainers in two dimensions and the sipper tube in one dimension) arealso possible.

The fluidic circuit 104 may include a valving module 106 that is capableof alternately directing fluid over the matrix in the first directionand back-eluting the elution fluid over the matrix in the seconddirection. Valving module 106 may include one or more valves. Forexample, FIGS. 2( a)-(c) are schematics of a chromatography system 200that includes a chromatography matrix 225 and two injection valves 206and 207, in accordance with one embodiment of the invention.

FIG. 2( a) shows the position of the valves 206 and 207 when the complexmixture is being loaded into a sample loop 208. A reduced pressure 221and an increased pressure 222 are continuously applied, by pumps, forexample, to a first port and a second of port of the valve 206. Thereduced pressure 221 is used to aspirate the complex mixture via asipper tube 204. The sipper tube 204 may be, without limitation,narrow-bore capillary tubing. Enough of the complex mixture to fillsample loop 208 with a defined volume is aspirated. The amount ofcomplex mixture to be passed over the matrix 225 can thus be controlledby the size of the sample loop 208. Any excess mixture aspirated iscollected in a trap 209 that may be positioned, for example, between theinjection valve 206 and the reduced pressure source 221.

In various embodiments, a wash solvent or buffer solution is positionedbetween the region of increased pressure 222 and the injection valve206. While the complex mixture is being loaded into the sample loop 206,the increased pressure 222 applied to valve 206 pumps wash fluid tovalve 207, which passes the wash fluid through the matrix 225 in a firstdirection 226. The output of the matrix 225 is diverted to waste by thevalve 207. Additionally, an increased pressure 223 continuously appliedto valve 207 pumps elution fluid, which may be positioned between theregion of increased pressure 223 and valve 207, to an analyzer 240. Inthis manner, carryover from previous complex mixture/samples is flushedfrom the matrix 225 and the analyzer 240 while the complex mixture isbeing loaded into the sample loop 206.

FIG. 2( b) shows the position of the valves 206 and 207 when the meteredcomplex mixture from the sample loop 206 is passed through the matrix225, in accordance with one embodiment of the invention. Upon actuationof the injection valve 206, the complex mixture, followed by wash fluid,is passed through the matrix 225 in the first direction 226. Due to thefocusing effect, the analyte of interest binds to the first part of thematrix 225, as discussed above. The wash fluid that follows the sampleensures sufficient removal of the undesirable components (e.g. salts,buffers, detergents, etc.) from the matrix 225, which are diverted towaste. To clean the sipper tube 204 prior to aspiration of the nextsample loop of complex mixture, the sipper tube 104 is dipped into awash solvent or buffer solution. The reduced pressure 221 applied to thesipper tube 104 passes wash solvent through the sipper tube 204 and intotrap 209.

After the analyte(s) of interest has been loaded onto the matrix 225 andthe undesirable components removed, the valves 206 and 207 divert thepumping system that loads the complex mixture onto the matrix 225 awayfrom the head of the matrix 225. Simultaneously, an elution fluid ispassed through the matrix 225 in substantially the opposite direction227 from which the complex mixture was loaded. The elution fluid, whichmay be either a solution or a solvent, dissociates the bound analyte(s)of interest from the matrix 225. In various embodiments, separatepumping systems are used to load the complex mixture, and pump theelution fluid across, the matrix 225.

FIG. 2( c) shows the position of the valves 206 and 207 when elutionfluid is back-eluted through the matrix 225, in accordance with oneembodiment of the invention. Valve 207 is actuated to pass the elutionfluid to the matrix 225 in the second direction 227. Since the analyteis primarily immobilized within the head 226 of the matrix 225 due tothe focusing effect, and does not have to travel the entire length ofthe matrix 225, thus limiting diffusion, the sample output of the matrixis delivered to the analyzer 240 in a concentrated manner within a smallbandwidth of time. While back-eluting, wash solution is passed throughthe sample loop 206 to clean and prepare the sample loop 206 forsubsequent aspiration of complex mixture.

The analyzer 240 may be, for example, an optical interrogator or massspectrometer. In various embodiments, the sample may be presenteddirectly to a mass spectrometer using a variety of standard systems,including atmospheric pressure chemical ionization (APCI), electrosprayionization (ESI) or atmospheric pressure photoionization (APPI). Themass spectrometer is capable of quantitatively analyzing a large numberof compounds based on the mass-to-charge ratio of each compound. Furtherseparation of individual compounds is generally not necessary, since anaccurate mass-selective detection and quantification can be performed bymass spectrometry. The output of the MS is analyzed and the amount ofcompound present in the sample is determined by integrating the areaunder the MS peak.

After back-eluting, both valves 206 and 207 are actuated as shown inFIG. 2( a). The steps of loading the complex mixture into the sampleloop 208 (if implemented), passing the complex mixture over the matrix225 in the first direction, and back-eluting the elution fluid over thematrix 225 in the second direction are then periodically repeated so asto achieve a high sample-throughput rate.

Minimizing Peak Width of Matrix Sample Output

The sample peak width (at half height) at the output of the matrix 225can be further minimized by selecting appropriate flow rates from thepumping systems 221, 222, and 223 and by selecting tubing diameters thatfurther minimize linear diffusion as the complex mixture and samples aremoved through the fluidic circuit 104. Typically, narrower bore tubingproduces sharper peaks enabling higher throughput, but also lead tohigher back-pressure in the fluidic pumping system. Similarly, higherflow rates also generally result in sharper peaks, but also lead tohigher back-pressure. High flow rates can also lead to decreased signalintensity in a mass spectrometer due to incomplete sample ionization.Determining the maximum throughput of the system is therefore acompromise between several factors that can be modeled or determinedempirically. The various parameters used, including the nature and typeof the insoluble matrix, pumping flow rates and pressures, tubingspecifications, the nature of the fluids used to perform the rapidchromatography, and the timing of the switching of the fluidic valves206 and 207 must be optimized for each family of chemical compounds tobe analyzed. This set of optimized parameters makes up acompound-specific method for high throughput mass spectrometricanalysis. In accordance with various embodiments of the invention,typical ranges for tubing diameters range from 20 μm to 300 μm and flowrates range from 0.1 mL/min to 5 mL/min resulting in backpressures thanmay reach anywhere from 5 to 6000 psi.

Minimizing Carryover

A major concern in maximizing sample throughput is the elimination ofsample-to-sample carryover. Referring back to FIG. 1, any sample that isnot removed from the fluidic circuit 104, matrix 102, and analyzerinterface 130 after one analysis may cause interference with the nextsample. If a sample with a low level of analyte is preceded by a samplewith a high level of analyte, carryover from the first sample may resultin an incorrect analysis in the second, low analyte sample. Minimizingcarryover is typically achieved by washing the fluidic circuit 104,matrix 102, and analyzer interface 130 with a solvent that fullysolubilizes the analytes of interest so that they are removed from thesystem 100. Various embodiments of the invention also use thistechnique, and the fluidic circuit 104, matrix 102, and analyzerinterface 130 may be flushed with the elution buffer/wash solution tominimize sample carryover.

Washing of the fluidic circuit 104 and other components of the system100 which contact the complex mixture and/or sample is conventionally atime consuming step and a long washing step between samples limits theoverall throughput of the system. Therefore, a system that requires aminimum amount of washing while producing an acceptably low level ofcarryover is highly desirable. This requirement can be achieved, inpart, by making those surfaces in the fluidic circuit 104 and othercomponents in the system 100 which contact the complex mixture and/orsample (including the sample loop 206, valving module 106, MS interface130, etc.) bio-inert so as to minimize the amount of carryover and easecleaning. Furthermore, due to the high backpressures generated by thepumping system, such surfaces must have a strong mechanical resistanceand the ability to resist high pressure liquids without leaks.

A commonly used material for such systems is Poly Ether Ether Ketone(PEEK) that has strong chemical resistance and can be manufactured in awide range of interior and exterior diameters. However, in preferredembodiments of the invention, the tubing within the fluidic circuit 104is manufactured from polyimide. Polyimide tubing has exceptionally lowcarryover of even very highly hydrophobic compounds, can resist highpressures before failing and can be manufactured in the 20-300micrometer inner diameters that are optimum for minimizing lineardiffusion. Use of a polyimide fluidic system allows for very rapidwashing steps between samples for a wide range of analytes with minimalcarryover. Another option for the construction of the fluidic circuit104 and other components which contact the complex mixture and/or sampleis titanium or titanium alloys that are also known to have low carryoverproperties. The fluidic circuit 104 may also include a microfluidicbiochip that may have, without limitation, channels having a diameterbetween 20 μm to 300 μm optimized for minimal linear diffusion.

Another embodiment of the invention is to construct the fluidic pathwayin full or in part from a material such as stainless steel. Stainlesssteel is not a particularly bio-inert substrate and tends to stronglyadsorb hydrophobic compounds in its surface. However, the surfaces ofthe fluidic circuit 104 and other components which contact the complexmixture and/or sample may be chemically or physically coated with ahydrophobic or hydrophilic film (e.g., Teflon, polyethylene glycol) bymethods known to those familiar to the art in a manner that willminimize the binding of analyte(s), thus minimizing carryover.

Fluidic Valves

In accordance with various embodiments of the invention, fluidic valvesin the fluidic circuit 104 are actuated to reverse direction of flowacross the chromatography matrix 102. Typically, the flow across thematrix 102 needs to be reversed twice for each sample output from thematrix 102. The complex mixture 102 is first loaded onto the matrix 102in one direction and the analyte(s) are bound but other components (e.g.salts, buffers, detergents, etc.) are not. The flow is then reversed andthe analyte(s) are eluted off of the matrix 102 in the oppositedirection to which there were loaded and diverted to the analyzer 116for analysis. Finally, the flow is reversed again in preparation for thenext sample. In many fluidic valves used in such microfluidicapplications, the flow of liquid through the valve is physically stoppedduring the time at which the valve is being actuated. Typicalelectronically actuated valve modules 106 can switch between states in100 milliseconds or slower. Pneumatically actuated valves may beswitched much faster, and may reach actuation times of 30 to 40milliseconds. This short blockage of flow during the actuation time isnot a concern during conventional LC where runs typically last minutes.

However, the blockage of flow becomes a concern at very high throughputrates where the sample throughput time approaches 1 sample/second.Typically, the injection valves that may be used in this system allowfor fluidic communication between two ports and have two actuationpositions. However, if the valve is adjusted to an intermediate statebetween the actuation positions, the fluid communication is physicallycut and no fluid can pass through the valve. During the actuationprocedure there is a finite amount of time as the valve is rotated fromone position to the other that the flow of fluid through the valve iscut. The high-pressure pumps that are pushing fluid through the valvescontinue to operate during this time. The creation of a blockage in flowat the valve during the actuation process results in an increase in thepressure within the fluidic circuit between the valve and thehigh-pressure pump. If the pressure increase is large enough it willeventually result in a failure of the fluidic system and could result ina leak. With conventional valves systems, the pressure increase istransient and the increase in pressure is not sufficient to actuallycause a failure of the fluidic circuit. However, the blockage in theflow will be observed in the baseline of the mass spectrometer signal.Since the impurities in the solvent that result in the background MSsignal are eliminated with a blockage in flow, the baseline tends todrop significantly during the valve actuation. When the valve hasfinished rotating and a fluidic connection is reestablished, theincreased pressure between the pump and the valve is released and ahigher than normal flow of solvent is delivered to the massspectrometer. This results in an increased amount of impurities enteringthe mass spectrometer and an increase in the background signal. If thisevent overlaps with analyte signal it can lead to unsymmetrical peaks,distorted baselines, and generally poor quantification.

Ideally, the reversal of flow occurs at such a speed that there is nodetectable disturbance in the flow rates and pressures during the flowswitching operation. A valve module 106 capable of 100 millisecondactuation times employed in an application where a sample throughput ofone sample per second is being performed means that the flow to theanalyzer 116 will be physically blocked for 200 milliseconds per second,or 20% of the overall sample analysis time. In various embodiments ofthe invention, a valve module 106 utilizing, without limitation,pneumatic valves capable of actuation speeds faster than 100milliseconds, and preferably on the order of 30 milliseconds or less areutilized.

Pneumatic valve actuators are available from VICI Valco Instruments ofHouston, Tex. Pneumatic valve actuators can be coupled to any valve(including valves from manufacturers other than VICO Valco Instruments)through a shaft coupling. Suitable valves include valves havingporcelain rotors and/or diamond-like coatings, such as NANOPEAK™ valvesavailable from Scivex, Inc. of Oak Harbor, Wis.

An additional advantage of the above-described embodiments overconventional systems is that the fluidic circuit is arranged such thatthe same solvent is always delivered to the mass spectrometer. Even whendoing a step elution, the elution solvent is the only solution that issprayed in to the mass spectrometer. While the wash solution containingthe mass spectrometer incompatible components of the reaction mixture isdiverted to waste, elution solvent is sprayed in to the massspectrometer inlet. In this manner a stable API spray is alwaysmaintained and variation in baseline due to different background signalsfrom wash and elution solutions is eliminated. Some advancedconventional systems divert the wash solution away from the MS inlet toavoid a buildup of non-volatile compounds in the source region. However,it can take several seconds to reestablish a stable spray in the MSinlet when the elution solvent is diverted to the MS. If the samplesignal overlaps with this region of unstable spray, it can lead toproblems with peak symmetry, baseline stability, and poorquantification.

Software

Software used to analyze the data generated by the analyzer 116, whichmay be executed by the controller 125 or another processor, enables manyfeatures of a high throughput analysis. For example, the massspectrometer output at the end of a long analysis at high throughputconsists of a series of data point in which time versus intensity valuesare recorded at each mass channel being analyzed. If plotted in aCartesian coordinate system, these graphs result in a chromatogram madeup of a series of peaks, wherein an integration of the area under eachpeak can be correlated to the concentration of the sample that wasanalyzed.

This integration event can be coupled to the switching of various valvesin the fluidic circuit 104, in accordance with one embodiment of theinvention. The time that a valve was actuated to back-elute the samplefrom the chromatography matrix into the mass spectrometer (or otheranalyzer) can be precisely recorded. It is known that until this eventtakes place no analyte(s) can be delivered to the mass spectrometer.Upon actuation of the valve, the mass spectrometer signal from theanalyte(s) being back eluted from the chromatography matrix can beobserved. The valve actuation time and the beginning of a massspectrometer peak can be accurately mapped to one another in time, suchthat the peak integration algorithm consists of an integration of themass spectrometer signal for a selected time period after each valveactuation. Even those samples that contain no detectable analyte(s) canbe accurately analyzed in this manner since an identical signal windowis monitored and integrated in each and every case.

In some cases, an error in the fluidic circuit 104 may lead to no signalbeing seen in the mass spectrometer. An example of such an error couldbe a fluidic reservoir in which no sample was present. This would leadto air being injected on to the column 225 rather than an aliquot ofsample. In such a case, only baseline signal would be detected for allanalytes. Since the final quantification relies on a relativemeasurement (i.e. substrate versus product or analyte versus an internalstandard) such an error can be easily detected. If the sum of the two ormore analyte signals is below a certain threshold, that sample can beflagged as an error.

Multiplexing

In various embodiments of the invention, the time required for rapidchromatography and inter-sample washing is much larger than the samplepeak width (at half-height) at the output of the matrix. In suchembodiments, there may be several seconds of baseline mass spectrometer(or other analyzer) signal before the next sample to be analyzed isdelivered to the mass spectrometer. This period is effectively a loss inproductivity, since the mass spectrometer is not actively quantifyingsamples.

Because mass spectrometers are large footprint instruments that requirea significant capital expense, two or more high throughput massspectrometry interfaces 303 and 304 are used to feed samples to a singlemass spectrometer 302, as shown in FIG. 3 in accordance with oneembodiment of the invention. Each mass spectrometry interface 303 and304 may include, without limitation, a rapid chromatograph system 100described above. A selection valve 310 is placed between the pluralityof high throughput mass spectrometry interfaces 303 and 304 and the massspectrometer 302. When a sample from a given high throughput massspectrometry interface 303 or 304 is ready to be analyzed, the selectionvalve 310 is used to direct that sample to the mass spectrometer 302while the remaining interfaces 303 or 304 are diverted to waste. Bystaggering the sample delivery to the mass spectrometer 302 such thatwhile one interface is being actively analyzed the others are in thewashing or sample acquisition steps, a plurality of interfaces 303 and304 can be used on a single mass spectrometer 302, allowing throughputto be maximized.

Auto-Injection Device

FIG. 4 is a schematic of an auto-injection device 400 that includes asingle injection valve 405, in accordance with one embodiment of theinvention. The auto-injection device 400 may be used in combination withadditional valves as in the above-described embodiment, and may be usedto transfer samples from a sample reservoir to, without limitation, afluidic circuit that may include an analyzer and/or a chromatographycolumn.

As shown in more detail in FIG. 5( a), and similar to valve 206 in FIGS.2( a-b), when the injection valve 405 is in a first position, (e.g. notactivated), a source of reduced pressure 411 is used to aspirate asample 401 through sample sipper tube 407 and into a sample loop 403.Upon actuation of the injection valve 405, the sample is introduced to afluidic circuit 413 by applying increased pressure, as shown in FIG. 5(b). To clean the sipper tube 407 prior to deactivation of valve 405 andaspiration of the next sample, the aspirator tube 405 may be dipped intoa wash solvent or buffer solution, with reduced pressure applied toaspirate wash solvent through the aspirator tube 104 and into trap 109.Thus, the combination of the constant negative pressure and the in-linetrap eliminates the need for repetitive aspiration and dispensing ofwash solution through a syringe.

Where an excess of sample is available, the reduced pressure source 411may be, without limitation, a vacuum pump that is capable of applying acontinuous vacuum to the distal end of the sample sipper tube 407. Whena large enough volume of sample 401 has been aspirated into the sampleloop 403 to fill it completely, the injection valve 405 is actuated andthe sample is output to the fluidic circuit 413. The trap 409, locatedbetween the injection valve 405 and the vacuum pump 411 is used tocollect excess sample. Changing the injection volume can be accomplishedby changing the length of the sample loop 403.

However, in cases where an excess of sample 401 is not available or thesample is too valuable to expend, a metered amount of sample 401 may beaspirated into the injection valve 405. In a preferred embodiment of theinjection, this metering is performed through the use of a cut-off valve415 located between the vacuum pump 411 and the injection valve 405. Inpreferred embodiments, the cutoff valve 415 is a solenoid valve withvery rapid response times allowing for accurate and precise actuation inthe millisecond time scale. The cutoff valve 415 may be used to aspiratean aliquot of sample into the sample loop 403 through the sipper tube407 for a very precise and controlled amount of time. The volume ofsample aspirated into the sample loop 403 can be precisely calibratedbased on the diameters of the sample loop 403, sample sipper tube 407,and the timing of the cutoff valve 415. The longer the cutoff valve 415is kept in the open position, the longer the aspiration of the sampleand the larger the volume of sample aspirated into the injection valve405.

In accordance with another embodiment of the invention, the continuousvacuum system may be replaced with a piston device in fluidcommunication with the injection valve 403, particularly in cases wherea cutoff valve 415 is impractical, or where the plurality of samples tobe analyzed has large differences in viscosity. Changes in viscosity maycause changes in the rate of sample aspiration. The amount of sampleaspirated into the sample loop 403 can be metered, for example, bycontrolling the distance the piston is withdrawn within a cylinder. Asufficient time is allocated to the aspiration process to permit theentire metered amount of sample to be loaded into the sample loop 403.Imprecision in injection volumes due to differences in rates ofaspiration caused by sample viscosity can thus be eliminated. The sampleis aspirated directly into the sample loop 403 and then injected intothe fluidic system. There is no need to apply positive pressure from thepiston until it has reached the end of its traverse within the cylinder.

In other embodiments of the invention, various combinations of theabove-described approaches for aspirating sample into the injection loopthrough the sipper tube may be used. For example, a selection valve maybe used to select whether a cutoff valve in combination with acontinuous vacuum source, or alternatively, a piston device, is placedin fluid communication with the injection valve. Where an excess ofsample is available, the selection valve is operated so as to place thecutoff valve and continuous vacuum in fluid communication with theinjection valve, with the cutoff valve left in the open position. If theaspiration of the sample must be metered either the cutoff valve can beactivated as described above, or the selection valve can be actuated touse the piston-based aspiration system.

The auto-injection device 400 is advantageous over conventionalauto-injectors for several reasons. By directly aspirating the sampleinto the injection loop rather than into a transfer syringe, thecomputer controlled robotic motion required for each injection isreduced. In conventional auto-injector systems, the transfer syringemust first be moved into the sample reservoir to aspirate an aliquot ofsample. Next the transfer syringe must be moved to the injection valveand the aliquot of sample loaded into the injection loop. After theinjection, the syringe must be moved yet another time to one or morecleaning stations. Because the current invention aspirates the sampledirectly into the injection loop, the need to move the transfer syringefrom the sample reservoir to the injection valve is eliminated.Minimizing robotic movement within the device both increases thethroughput and the reliability of the system. By repeatedly aspiratingand injecting from the same sample, larger sample volumes may beanalyzed without undue delay. If the injection is to a chromatographicresin, multiple aliquots of sample may be added to the column prior toadditional steps of washing and eluting.

Another advantage of the current invention is realized in the cleaningof the auto-injector between samples. All surfaces that come intocontact with sample generally must be thoroughly cleaned before the nextsample can be injected. In conventional auto-injectors, this includesthe injection valve as well as the transfer syringe. Cleaning of thetransfer syringe can be especially challenging and time consuming,especially if a standard syringe is used. Because most syringes aremanufactured from glass and stainless steel, certain samples areparticularly difficult to remove. Many lipophilic compounds tend toadhere strongly to stainless steel and can lead to sample carryover orleaching. Transfer syringes typically have tubing of various diametersand are composed of multiple materials (e.g., glass and stainless steel)that are more difficult to clean than continuous and smooth bio-inerttubing. Since the current invention aspirates samples directly into theinjection valve through a sipper tube, the invention does not requirethe cleaning of a transfer syringe. This has the double impact ofdecreasing sample carryover while also increasing the throughput of thedevice.

Of importance to minimizing sample carryover is the choice of materialused for the sipper tube. In a preferred embodiment of the invention, aconcentric tube injector is used to provide the ability to pierce sealedsample reservoirs without the need to have the sample come into contactwith materials such as stainless steel that are not chemicallycompatible with a wide range of samples, as described in U.S. Pat. No.7,100,460.

As described above, cleanup of the device can be achieved by simplyaspirating a large volume of fluid through the sipper tube 407 while thesample of interest is being diverted to the fluidic circuit foranalysis, as shown in FIG. 5( b). The use of biocompatible materialscoupled with the small surface area of the sipper tube and injectionvalve that needs to be cleaned allows for very efficient reduction ofsample carryover while maintaining a rapid throughput.

The following are examples, without limitation, of high-throughputsampling using various configurations of the above-describedembodiments.

Example 1 Drug-Drug Interaction (DDI) Assay

Many xenobiotic compounds are metabolized in vivo by a family of enzymesknown as cytochrome P450s, primarily in the liver. The metabolicactivity by P450 enzyme also includes a vast majority of small moleculepharmaceuticals. Since the therapeutic activity of many pharmaceuticallyactive compounds is highly dose-dependent it is advantageous tounderstand the metabolic fate of these chemicals. In many cases, highdoses of certain chemicals can be toxic or have long-term deleteriouseffects.

Many compounds are known to affect the metabolism of certain P450enzymes, either acting as inhibitors or activators. This is true for arange of chemicals that are currently used as therapeutics. It iscritical to know from a drug-safety standpoint whether or not themetabolic profile of a pharmaceutical compound taken by an individualmay be affected by other chemicals that individual may be taking. If anindividual is currently taking a certain drug that inhibits the actionof a specific P450 enzyme, taking a second drug that is also metabolizedby the same P450 enzyme can have catastrophic consequences. Theinhibitory effect of the first drug on the P450 enzyme can lead to thesecond drug not being metabolized at the predicted rate and result inmuch higher than expected in vivo concentrations. In some cases this canbe toxic or even fatal.

To study the possible effects of potential new pharmaceutical compoundsa series of in vitro assays known as the drug-drug interaction assayshave been developed and are familiar to those skilled in the art. Theassays use various preparations of P450 enzymes, either as purifiedrecombinant proteins, or as various cellular or sub-cellular (e.g.microsomes, S9 fractions, etc.) preparations of liver tissue. The enzymepreparations are allowed to react with known substrates of the P450enzymes, known as probes, under controlled conditions in the presence ofthe test compounds. If the test compound is active in the assay it willcause a shift in the expected metabolism of the probe molecule. A widerange of different probes and assays has been described in thescientific literature. These formats include both optically activeprobes typically used with recombinant enzyme preparations and massspectrometric approaches that facilitate the use of subcellular liverpreparations and highly selective and specific probes. While thethroughput of optical assays can be very high, researchers generallyprefer to perform mass spectrometry-based assays since more biologicallyrelevant data can be obtained.

The above-described embodiments of the invention can be used to vastlyimprove the throughput of mass spectrometry-based drug-drug interactionassays. An assay to test the activity of test compounds againstcytochrome P450-2D6 (CYP2D6) was performed in a 96-well microtiterplate. A microsomal preparation from human liver tissue was incubated inthe presence of dextromethorphan, the test compound, and NADPH in abuffer containing potassium phosphate at pH 7.4 and magnesium chloride.After a 30 minute incubation, the reaction was quenched by acidifyingthe reaction with the addition of 10% (v/v) 0.1% formic acid. While thehuman liver microsomes have a variety of different enzymes,dextromethorphan is a specific substrate of CYP2D6 and is metabolizedinto dextrorphan. While the remaining dextromethorphan substrate and thedextrorphan product formed in the reaction can be quantified by the useof conventional liquid chromatography-mass spectrometry at throughputsthat is on the order of minutes per sample, the above-describedembodiments of the invention allow similar analysis to be performed onthe order of 5 seconds.

In accordance with an embodiment of the invention, and referring toFIGS. 2( a)-(c), the sipper tube 204 attached to the valve 206 is movedrelative to the first sample to be analyzed in the 96-well microtiterplate. The distal end of the sipper 204 is immersed into the reactionbuffer and an aliquot is aspirated into a 5.0 microliter injection loop208 through the use of a vacuum 221 applied to the distal end of thesipper tube 204. Fifty milliseconds after aspiration has begun, enoughfluid has been aspirated into the valve 206 to completely fill the 5.0microliter injection loop 208. At this time the injection valve 206 isactuated and the sample in the loop 208 is brought into fluidcommunication with the output from a high-pressure fluidic pump 222 thatpushes the 5.0 microliter sample aliquot through the injection loop 208and onto a chromatographic column 225 containing an insoluble matrix.The matrix consists of impermeable beads that are an average of 40microns in diameter. The surface of each bead has been derivatized witha 4-carbon long alkane chain to create a hydrophobic environment. Porousfrits constrain the insoluble matrix beads within the column 225,however, the nature of the particles allows for fluid to freely movearound and between the particles without an unacceptably high increasein pressure.

The high-pressure fluidic pump 222 is used to pump water at a flow rateof 1.2 milliliters per minute. When the sample reaches the column 225the dextrorphan and dextromethorphan analytes, being lipophilicmolecules, interact with the insoluble matrix beads within the columnand are adsorbed onto the column 225. Compounds in the reaction bufferthat interfere with mass spectrometry, including the potassium phosphatebuffer, magnesium chloride salts, NADPH and NADP, are highlyhydrophilic, and accordingly, are flushed through the column into awaste container. Insoluble components in the assay buffer that may havebeen aspirated along with the sample are small enough to move throughthe space between the 40 micron beads and are also removed from theanalytes.

The total internal volume of the column 225 is 4.0 microliters. Toremove the interfering salts at an acceptable level it is necessary toflush the column with several volumes of water. At a flow rate of 1.2milliliters per minute, a total of 20 microliters of water per second ispumped. Therefore in the one-second wash, a total of 5 column volumes ofwater were pumped over the bed of matrix to remove the mass spectrometryincompatible components.

During this entire process a second fluidic pump 223 is used to pump asolution of 80% acetonitrile in water at a flow rate of 1.0 millilitersper minute directly on to a triple quadrupole mass spectrometer 240operating in the electrospray ionization (ESI) mode. The massspectrometer 240 was optimized to specifically monitor the dextrorphanand dextromethorphan analytes in multiple reaction monitoring (MRM)mode. A stable ESI flow was maintained and a constant baseline from the80% acetonitrile solution was established. Exactly 1.0 seconds after thevalve 222 to push the sample from the injection loop onto the matrix,the second valve 207 was actuated. This valve 207 forces the 80%acetonitrile to enter the column 225 from the direction opposite thatfrom which the sample was loaded. Simultaneously, the output of thefirst pump 222 was diverted away from the column and to waste. Thesecond valve 207 actuation brought the column 225 into fluidic contactwith the second pump 223 and the analytes adsorbed on the column 225were eluted by the 80% acetonitrile and pushed into the ESI source ofthe mass spectrometer 240 where they are analyzed. The two analytes wereeluted simultaneously and analyzed in the mass spectrometer based ontheir mass to charge ratios.

Since the elution step is done in the opposite direction of the loadingstep, the analytes never travel through the column 225. This is animportant point, since fluid traveling over a column 225 tends toundergo turbulence that can result in mixing and linear diffusion.Minimizing the linear diffusion is very important since this leads to anincrease in the volume of fluid in which the sample is presented to themass spectrometer. Best analytical data is obtained when the sample ispresented in the smallest possible elution volume in the shortest amountof time. Small elution volumes lead to high local concentrations ofanalyte and a correspondingly high signal level that can bedistinguished from the background signal and shot noise. In 1.5 secondsat a flow rate of 1.0 milliliters per minute a total of 25 microlitersof elution fluid was pushed over the column 224. This corresponds toover 6 column volumes of elution fluid, more than enough to flush thecolumn and eliminate carryover to the next sample.

At this time both valves 222 and 207 were actuated again to theirstarting positions. The injection loop 208 was available to aspirate thenext sample, the 80% acetonitrile from the second high pressure pump 223was diverted away from the column 225 and directly to the massspectrometer 240 and the water from the first high pressure pump 222 waspushed over the column 225 in the original direction. This state wasmaintained for a minimum of 2 column volumes to (a minimum of 400milliseconds) to allow the local environment within the matrix of thecolumn 225 to be flushed with water to allow binding of the analytes inthe next sample. This process is known as column equilibration and mustbe performed in sufficient time to allow proper analysis. After theequilibration of the column 225 the next sample was ready for analysis.

While the sample was being analyzed 80% acetonitrile from a reservoirwas aspirated through the sipper tube 204 to remove any contamination inthe sipper tube and to eliminate carryover into the next sample. In thismanner, samples were analyzed at a periodic rate of 5 seconds persample. It is also possible to analyze more than two analytessimultaneously and therefore to multiplex assays for multiple P450isoforms.

Example 2 Metabolic Stability Assay

In accordance with various embodiments of the invention, certainattributes of the system may be advantageously enhanced at the expenseof throughput. An example of such an application is the metabolicstability assay. The metabolic stability assay assesses the activity ofliver enzymes on a test molecule. It is typically an in vitro assayperformed by incubating a sub-cellular liver preparation (e.g. livermicrosomes or S9 fraction) with a source of energy (e.g. NADPH) and thetest compound in an appropriate buffer system. The liver enzymes maymetabolize the test compound, the rate of which can be determined byquantifying the amount of the test compound at controlled times usingmass spectrometry.

This assay is different from the DDI assay in that each and every testcompound must be monitored in the mass spectrometer. In the DDI assay,only a specific set of probes needed to be monitored allowing for a fulloptimization of the system. Given that a very wide range of testcompounds needs to be analyzed in the metabolic stability assay, genericmethods capable of analyzing many different chemical structures arerequired. In this application, the throughput of the system is loweredslightly to facilitate the analysis of a wider range of analytes.

To perform the metabolic stability assay, a different approach than theDDI assay is used. The reverse elution (i.e., eluting the analytes fromthe column in the opposite direction to which it was loaded onto thecolumn) is not used. Rather, the analytes are eluted from the column inthe same direction as they were loaded on to the column. This results inlinear diffusion as the analytes experience turbulent flow as they arepushed over the insoluble matrix beads, causing a broader peak andtherefore lower throughput. However, many of the aspects of theinvention used in the DDI assay can still be applied to the assay andresult in a significantly increased throughput over conventional methodswithout a sacrifice in the sensitivity of the assay. These advantageswill be described in detail below.

The sample aliquot is aspirated into an injection loop and loaded ontothe column in the same manner as in the DDI assay with a firsthigh-pressure pump that is used to pump a wash solution. This solutionis typically water or an aqueous buffer and is used to flush out thesalts, buffer components, NADPH, and insoluble components of thereaction mixture to a waste container. During this time a second highpressure pump is used to pump an elution solvent in to the ESI or APCIsource of the mass spectrometer. When the second valve is actuated, theelution fluid is forced over the column in the same direction that theanalytes were loaded on to the column. An elution fluid that is capableof dissolving a very wide range of chemicals but is compatible withatmospheric pressure ionization mass spectrometry is used. These buffersmay include alcohols (e.g. methanol, ethanol, or isopropanol),acetonitrile, acetone, tetrahydrofuran or mixtures of these solvents. Itis generally desirable to have a small amount of water in the mixture,and additives such as ammonium acetate, ammonium carbonate, or DMSO tothe elution solution may result in sharper peaks.

If the mass spectrometric characteristics of the analytes of interestare previously known, the mass spectrometer can be set up tospecifically monitor those compounds in MRM mode. However, if noprevious information is available about the analytes, it may bedesirable to use a mass spectrometer to scan a range of masses. The useof a time-of-flight, ion trap, hybrid quadrupole/ion trap, or hybridquadrupole/time-of-flight mass spectrometer can facilitate the scanningof a wide range of masses with minimal loss in signal intensity. Toobtain good quantitative data an internal standard is added upon thequenching of the reaction and the signal from the analyte(s) isnormalized with respect to the internal standard.

The current invention uses a step-elution system to purify samples usingcolumn chromatography and analyses them using mass spectrometry.However, the system uses a significant improvement over conventionalstep elution systems: the same solvent system (the elution solution) isalways sprayed into the inlet of the mass spectrometer. In conventionalsystems, as the wash and elution solutions are alternated for eachsample and the two different solutions are alternately sprayed in to themass spectrometer inlet. This can have a huge impact on the baselinesignal observed. The variation is baseline signal may have a significantimpact on the quantification of peaks, particularly those that have alow level of signal.

In some of the more advanced conventional systems the wash solution isdiverted away from the mass spectrometer inlet to a waste container andonly the elution solution is sprayed into the mass spectrometer.However, this also results in a change in the background signal seen inthe mass spectrometer since there is no flow during the column loadingand washing stages. Furthermore it may take several seconds toreestablish a stable spray in the MS inlet. In a high throughput systemsuch as described here, the leading edge of the analyte peak may overlapwith the region of unstable flow resulting in poor sensitivity, unevenpeak shape and increased error in quantification.

A further improvement the current invention provides over conventionalsystems is in the fast switching valves. Typical electronically actuatedvalves provide switching times over 100 milliseconds. However the veryfast valve switching (e.g. 50 milliseconds or less) employed in thecurrent invention allows for a pulse-free spray in the mass spectrometerproviding symmetrical peaks with flat baselines and facilitates accuratequantification.

Example 3 Compound Purity Testing

In some applications, the samples to be analyzed are already in a bufferthat is compatible with mass spectrometry. Such an application may bethe quality control analysis of test compounds in an aqueous or organicbuffer that can directly be sprayed in to an API source without the needfor any purification. Various aspects of the above-described embodimentscan be used to increase the sample throughput for such an application.

In this application only a single injection valve is used. An aliquot ofsample is aspirated into the injection loop and upon actuation of thatvalve the sample is sprayed directly in to the mass spectrometer. Theflow from a single fluidic pump is used to push the sample through theinjection loop and into the inlet of the mass spectrometer. In preferredembodiments of the invention, the fluid used to push the sample on tothe mass spectrometer is one that the analytes are highly soluble in andprovides good ionization in the mass spectrometer inlet. These solutionsmay include alcohols (e.g. methanol, ethanol, or isopropanol),acetonitrile, acetone, tetrahydrofuran or mixtures of one or more ofthese solvents with water.

The system provides increased throughput over conventional systemsthrough several means. The elimination of a transport syringe to move asample aliquot from a reservoir to the injection valve increases theoverall speed of the robotics. The injection valve and the samplereservoir may be moved relative to each other such that an aliquot ofsample can be directly aspirated in to the injection loop. Besidesfacilitating faster robotics, this also eliminates the need to clean thetransport syringe between injections. In conventional systems both thetransport syringe and the injection loop need to be thoroughly cleanedbetween samples. However, in the current invention there is no transportsyringe.

Samples that do not need to be purified have been analyzed byatmospheric pressure ionization at throughputs of approximately 1 secondper sample using the system with minimal carryover between samples.

Avoiding Sample-To-Sample Carryover

As explained above, the entire fluidic system is properly cleanedbetween analyses to ensure that carryover from a given sample does notconfound the analysis of the next sample in the cue. Cleaning of thefluidic system is achieved by flushing the fluidic system with a solventthat the confounding compounds are freely soluble in. The fluidic systemdescribed herein is composed of two major components, each associatedwith a valve assembly. A first valve contains an injection loop and asample aspiration tube through which an aliquot of a fluidic sample tobe analyzed is aspirated into the injection loop. This valve arrangementis in fluid communication with the second valve that contains achromatography system containing an insoluble matrix that is capable ofpurifying the sample prior to analysis.

Samples aspirated into the injection loop are loaded onto thechromatography column and washed with a “wash solution” in a firstdirection and, after an appropriate purification has been performed, thesamples are eluted from the chromatography column with an elutionsolution in a second direction, opposite to the first direction. Sincethe elution solution removes the sample from the column by solubilizingit, it has the secondary effect of cleaning the chromatography columnand effectively reducing carryover into the next sample. Several columnvolumes of elution solution may be necessary to reduce carryover to anacceptable level, depending on the exact nature of the analyte ofinterest and elution solvent used. A larger volume of elution solventcan be delivered to the column by increasing the time that the column isflushed with the elution solution in the second direction.

While the action of eluting the analyte off of the chromatography systemhas the added benefit of cleaning the column and valve assembly,carryover can also result from traces of analyte left in the injectionloop, sample aspiration tube, or in the portions of initial valve. Thisportion of the fluidic circuit can also be flushed with elution solutionto clean the system between samples and eliminate or minimize carryovereffects. In one embodiment of the invention, the sample aspiration tubecan be moved to a reservoir containing elution solvent. The valve can beactuated such that the sample loop is in fluid communication with thesample aspiration tube, and a necessary volume of elution buffer can beaspirated through the sample aspiration tube and the injection loop. Thesolvent so aspirated will be collected in the in-line trap downstream ofthe valve before the source of vacuum.

In one representative embodiment, the aspiration of the elution solventthrough the sample aspiration tube and injection loop will occursimultaneously with the back elution of the analyte from thechromatography column to the analyzer. This allows maximizing of thethroughput since the flushing of the first valve containing the sampleinjection loop will be achieved during the time that the sample is beingeluted. However, there may be particularly difficult analytes (e.g. veryhydrophobic compounds) that despite the use of bioinert materials andsurface coatings still cause carryover to be observed. Theseparticularly difficult analytes may require a large volume elutionsolvent to be aspirated through the sample aspiration tube and theinjection loop to eliminate carryover to an acceptable level. In somecases the aspiration of a large volume of elution solution through thesample aspiration tube and the injection loop may take longer than theback-elution of the analytes from the chromatography column to theanalyzer. In such a case flushing the fluidic circuit to minimizecarryover becomes a limiting factor for system throughput.

Thus another embodiment of the invention addresses the case of thoseanalytes where flushing the fluidic system is a throughput-limitingevent. Such an embodiment increases the volume of elution solvent withwhich a portion of the fluidic system is flushed between samples tofurther minimize carryover while still maximizing sample throughput. Tothis end, an additional fluidic valve can be used. This valve can be aselection valve or a 4-port injection valve. In one embodiment, a 6-portinjection valve is used which is identical to the two existing valveswhere 2 of the ports have been short-circuited with a piece of tubing.The use of identical valves throughout the fluidic circuit hasadvantages in manufacturing, inventory management, and servicing of theinstrument.

The fluidic valve associated with the sample injection loop (valve 1) isflushed with elution fluid from a positive pressure source, such as anadditional high-pressure fluidic pump, rather than being aspiratedthrough the valve with a vacuum as described above. The use of positivepressure allows for a much larger volume of fluid to be flushed throughthe valve in a given amount of time as compared to the use of vacuumaspiration. Under normal conditions, the maximum pressure with which afluid can be aspirated is 1 atmosphere, assuming a perfect vacuum can beapplied. In comparison, a standard high-pressure pump can apply tens ofatmospheres of fluidic pressure, resulting in a much larger volume offluid being delivered in an equivalent amount of time.

In this embodiment, the sample is aspirated into the sample loop onvalve 1 as before. The valve is then actuated and the analytes arediverted to the second valve and loaded onto the chromatography columnas before. Once the sample is purified through flushing with anappropriate volume of wash solution, valve 2 is actuated and the sampleis back-eluted onto the analyzer with elution solution. During this timethe additional upstream valve is simultaneously actuated to flush thefluidic circuit in valve 1 (including the sample injection loop) withelution solution. The sample aspiration tube is not in fluidcommunication with the sample loop at this time, and still must beflushed with aspiration of elution solution from an appropriatereservoir. Before the next sample is aspirated the additional valve isactuated once again and wash solution is flushed through the fluidicsystem and the chromatography column to equilibrate the system.

Three-Valve Embodiment

FIGS. 6(A)-(D) show an embodiment of the present invention using athree-valve and three-pump arrangement as described above. FIG. 6(A)shows the first assay phase in which a liquid sample is loaded into thesample injection loop. A sipper tube 604 is lowered into the samplereservoir and an aliquot of sample is aspirated into the injection loopvia sample injection valve 601 much as described before with respect totwo-valve embodiments. Any excess sample aspirated is collected in avacuum trap 621 downstream of the loop. During this time fresh elutionsolvent is delivered to the analyzer 640 from first elution solvent pump624 to establish and maintain stable ESI or APCI spray and acorresponding stable baseline signal from the mass spectrometer. Thecolumn 625 is equilibrated by pumping wash buffer (typically an aqueoussolution) from wash solvent pump 622. Second elution solvent pump 623 isdiverted directly to waste by solvent valve 603.

FIG. 6(B) shows the next phase, in which the valves are aligned to loadthe sample onto the column 625 and wash the lines. Sample injectionvalve 601 is actuated and the sipper tube 604 is raised out of thesample reservoir. The sample aspirated into the injection loop isdelivered to the column 625 where the analytes of interest bind, butinterfering compounds (e.g., salts, detergents, etc.) pass over thecolumn 625 and are sent to waste. An appropriate number of columnvolumes of wash buffer are pumped to over the column 625 to ensure thatthe sample is purified properly.

Next, as shown in FIG. 6(C), the sample is back eluted off the column625 into the analyzer 640. Column control valve 602 and wash controlvalve 603 are simultaneously actuated while the sipper tube 604 is movedinto the “wash solution” reservoir. The wash solution is aspiratedthrough the sipper tube 604 to clean it and eliminate sample-to-samplecarryover. Actuation of column control valve 602 results in the elutionsolvent from first elution pump 624 to be delivered to the column 625 inthe opposite direction and the bound sample is back-eluted into theanalyzer 640. An appropriate number of column volumes are pumped overthe column 625 to fully elute the sample. Actuation of wash controlvalve 603 causes elution solvent to be pumped by second elution pump 623over the injection loop and diverted to waste. This allows for acomplete flushing of the injection loop with elution solvent betweeneach sample and helps to minimize carryover.

Finally, FIG. 6(D) shows equilibration of the column 625 and aspirationof the next sample. All three valves 601, 602 and 603 are simultaneouslyactuated to their home positions. Fresh elution solvent is pumped to theanalyzer 640 from first elution solvent pump 624, while wash solvent ispumped over the column 625 in the forward direction. An appropriatenumber of column volumes are pumped over the column 625 to ensure columnequilibration. The sipper tube 604 is then dipped into the next sample,an aliquot is aspirated into the injection loop, and the cycle isrepeated.

The term “column volume” is referred to several times. A variety ofcolumn geometries, volumes, and packing can be used in various specificapplications and may be optimized for each application. Both physicalcharacteristics of the packing material (i.e., particle size, shape,porosity, etc.) and packing chemistry (i.e., C-18 vs. polymericpackings, etc.) are important in optimizing a give embodiment.Typically, column bed volumes under 10 μL are used. At a flow rate of1.2 mL/min, 20 μL/second is delivered to the column 625. This means thatin a typical 1.5 second elution, 3 volumes of a 10 μL column or 6volumes of a 5 μL column can be realized. Maximum throughput is achievedby using the smallest acceptable column bed volume for a givenapplication.

The elution solvent (usually an organic solvent) is delivered to theanalyzer 640 in an uninterrupted manner at a constant flow rate. Eventhough a step elution is performed, only the elution solvent isdelivered to the analyzer 640 in this arrangement. This allows for astable ESI or APCI flow to be established and a constant baseline to beachieved. Analytes are simply “inserted” into this flow with theactuation of column control valve 602 facilitating very sharp andsymmetrical peaks on a constant and stable baseline.

Other methods for performing a fast-step elution may be somewhatproblematic. For example, the wash solution and elution solvent can bedelivered to the analyzer 640 in a periodic manner. But this can resultin large changes in background signal from the two solutions. Attemptingto deconvolute a peak on a changing baseline is very troublesome. Also,the wash solution (typically containing the salts and other incompatiblecomponents of the sample mixture) can be diverted to waste and only theelution solution diverted to the analyzer 640. But establishing a stableESI flow can take significant time and if the analytes of interest aredelivered to the analyzer 640 before a stable flow is established,sensitivity and quantification issues are encountered.

Standard electro-mechanically activated valves may not be appropriate inthe embodiments just described due to slow actuation times. Since thevalves do not permit fluid communication during the actuation process,flow is physically cut. The interruption in flow manifests itself as a“negative” peak in the baseline signal twice per cycle (actuation thevalve and then returning to the home position). Given the throughputs ofspecific embodiments, at least one of these two “negative” peaksinterferes with the analyte signal from the analyzer 640 resulting in adouble peak that impacts on quantification and data quality. Thesolution is a very fast actuating valve (e.g., 30 μsec actuation time orfaster) which has been engineered specifically for the task. Theactuation time of the valves should be rapid enough that no interruptionto the flow can be observed physically or in the data.

Wash control valve 603 can be replaced with a 4-port valve. However, tokeep manufacturing and parts inventory simple, a six-port valveidentical to valves 601 and 602 may be preferred, and 2 ports can bepermanently “short-circuited” with a piece of tubing. To reduce samplecarryover and promote valve performance and durability, various valvecomponents (e.g. the stator) can be formed from ceramics and can becoated with materials such as polytetrafluoroethylene (PTFE) ordiamond-like carbon (DLC). Suitable valves include part number S-15287available from Upchurch Scientific, Inc. of Oak Harbor, Wash.

Wash control valve 603 also could be eliminated from the system and theinjection loop could be cleaned between samples by placing the sippertube 604 into the wash solution and aspirating solvent through thesample supply valve 601 in the “load” position where the injection loopis in fluid communication with the sipper tube 604. However, a greateramount of fluid can be pumped with positive pressure than can beaspirated with negative pressure in a given amount of time. For analyteswhere sample-to-sample carryover is problematic, the use of wash controlvalve 603 can help minimize carryover by washing the loop with a greateramount of solvent while maintaining throughput.

The valves can be actuated by an electric or pneumatic actuation.Suitable actuators include the VEXTA® two-phase stepping motor,available from Oriental Motor Co., Ltd. of Tokyo, Japan under partnumber P0040-9212KE. The actuator and the valve can be coupled by partnumber DK/GS 9 from GERWAH Drive Components, LP of Fayetteville, Ga.

In certain embodiments, the valve actuator(s) is controlled withsoftware and/or hardware. The software and/or hardware can control thetiming of movement of the valve actuator(s). Additionally, the softwareand/or hardware can control the velocity and/or acceleration of thevalves to achieve optimal performance and/or longevity. For example, inhigh-speed application, it is desirable to apply a braking ordecelerating force as the valve approaches the desired position in orderto prevent damage to the valve.

FIG. 6(E) depicts an embodiment of the three-valve embodiment. System600 includes valves 601, 602, and 603 (not visible), and sipper 604, allmounted on a bracket 650. The valves 601, 602, 603 are connected bytubing as depicted in FIG. 6( a). Advantageously, by mounting the valves601, 602, 603, and sipper 604 on the bracket, the length of tubingrequired is minimized, which enables higher throughput as smallervolumes are held in the tubing.

Each motor 651, 652, and 653 (not visible) is also mounted on bracket650 for actuation of valves 601, 602, and 603. In some embodiments, thesipper 604 is a sipper in accordance with U.S. Pat. No. 7,100,460.

The bracket 650, along with the sipper 605 can be moved at leastvertically (i.e. in the y direction) by control assembly 654, which canbe an electrical, mechanical, or electromechanical device as known tothose of skill in the art. Likewise, reservoir plate 401 can be moved atleast in the x and z directions by control assembly 655, which can be anelectrical, mechanical, or electromechanical device as known to those ofskill in the art. Control device 655 can interact with plate handlingdevice 656 to obtain pre-loaded plates 401 and return plates aftersamples are extracted.

FIG. 6(F) provides an isometric projection of bracket 650, with valves601, 602, 603 (again, not visible), motors 651, 652, and 653, and sipper604. As seen more clearly in FIG. 6(E), valves 601, 602, and 603 aremounted at angles so that the tubing distance between each valve can befurther reduced.

When used in conjunction with other aspects of this invention, such asthe use of bio-inert materials and surface coatings, three valveembodiments result in a minimum amount of carryover for even the mostdifficult compounds, while allowing for a very high rate of sampleanalysis.

Improved Fluid Injection Valve Timing

Another embodiment of the present invention provides a device and methodfor the rapid sequential analysis of a plurality of samples. The devicecomprises a computer controlled robotic system that aspirates an aliquotof fluidic sample from a sample reservoir directly into an injectionloop in a fluidic injection valve. This improvement allows for thedevice in the current injection to realize higher throughputs whileminimizing sample carryover.

As depicted in FIG. 7, activation of the valve is controlled by afeedback mechanism that includes a fluidic sensor 702 located betweeninjection valve 405 and vacuum trap 409. Fluid sensor 702 detects thepresence of fluid in conduit 704. The fluid sensor 702 is used tocontrol the precise timing at which the injection valve 405 is actuatedas well as determining the position of the sample aspiration tube 407with respect to the fluidic sample to be analyzed. Several embodimentsof the invention are described below.

In accordance with one aspect of the invention, the transfer syringe iscompletely eliminated from the auto-injection device. The sample to beanalyzed is aspirated directly in to the injection loop 403 of theinjection valve 405 through an aspiration tube 407 attached directly toa port of the injection valve 405. The computer-controlled roboticsystem of the device allows for the movement of the aspiration tube 407connected to the injection valve 405 to be moved relative to the samplereservoirs 401.

An aliquot of sample can be aspirated directly into the injection valve405 through the aspiration tube 407 in a number of ways. In cases wherean excess of sample is available and continuous vacuum may be applied tothe distal end of the injection valve 405 with a vacuum pump 211. When alarge enough volume of sample has been aspirated into the injection loop403 to fill it completely the injection valve 405 is actuated and thesample is introduced to the fluidic circuit. A trap 409 located betweenthe injection valve 405 and the vacuum pump 211 is used to collectexcess sample. In this embodiment of the invention, the amount of sampleinjected is controlled solely through the volume of the injection loop403. Changing the injection volume requires changing the injection loopof the device.

During sample analysis it is important that the injection volume isknown such that an accurate measurement is achieved. This isparticularly important when a plurality of samples is analyzed serially,since an inconsistent injection volume may lead to variability in themeasurements. The current invention relates to a device and method thatensures that a full injection loop with a minimum amount of sample isachieved with every sample aspiration, even in cases where theviscosity, amount, temperature, or other physical parameters of aplurality of samples may differ.

As the aspiration tube 407 is lowered into the fluidic sample 401, theaspiration tube 407, injection loop 403, and valve 405 will be free ofliquid and contain only ambient air. When the aspiration tube 407 isdipped into the fluidic sample 401, the sample 401 will be drawn intothe aspiration tube 407 and fill the injection loop 403 and eventuallybe collected within a vacuum trap 409. Actuation of the injection valve405 results in the volume of fluidic sample located within the injectionloop 403 to be introduced into the sample purification or analysisdevice. The timing of this actuation is critical since actuation of thevalve 405 too quickly results in an incompletely full injection loop 403while actuation of the valve 405 too late results in a waste of sample.In cases where only a small amount of fluidic sample is available foranalysis, a late valve actuation may lead to the entire sample travelingthrough the injection loop 403 and being collected in the vacuum trap409, resulting in an incomplete or empty injection loop 403 and a lossof the sample.

While it is possible to determine the timing of the proper valveactuation empirically, this is a time consuming and error-prone processthat typically results in an excess of sample being aspirated leading towaste. Empirical determination of the valve actuation timing becomesexceedingly difficult when the volume of the fluidic sample to beanalyzed is very small. Furthermore, small changes in the physicalcharacteristics of the fluidic sample, such as the viscosity,temperature, or insoluble materials such as cellular or sub-cellularcomponents can greatly affect the sample aspiration rate leading toinconsistency when a plurality of samples is aspirated. The presentinvention overcomes these problems.

The present invention provides a device wherein activation of injectionvalve 405 is controlled by sensor 702. In some embodiments, the sensor702 also controls certain aspects of the sample aspiration process.

In one embodiment, the device of the current invention comprises asensor 702 that is placed between the injection loop 403 and the vacuumsource 211. When fluidic sample reaches the sensor 702 the interfacebetween air and fluid generates a signal that is used to trigger theactuation of the injection valve 405. The presence of fluid at thesensor 702 can only be achieved when the sample aspiration tube 407 andinjection loop 403 are completely full of fluidic sample. By minimizingthe volume between the distal end of the injection loop 403 and sensor702 the amount of sample that is aspirated prior to the actuation of thevalve 405 can be minimized, resulting in a minimal amount of samplebeing wasted during the analysis.

In another preferred embodiment of the present invention, feedback fromthe sensor 702 is used not only to trigger the actuation of theinjection valve 405 when the sample injection loop 403 is full, but alsoto control the mechanical movement of the sample aspiration tube 407. Inthis embodiment, as depicted in FIGS. 8( a)-(c), the sample aspirationtube 407 is lowered into the vessel 802 in which the fluidic sample tobe analyzed is contained. The level of fluid within this vessel 802 doesnot need to be known a priori. When the aspiration tube 407 is moved toa position below the level of the fluidic sample (FIG. 8( b)), thesample will be aspirated through the aspiration tube 407 into the loop403. When the loop 403 is full and the fluidic sample reaches andtriggers the sensor 702, the injection valve 405 will be actuated andthe sample aspiration tube 407 will be moved such that it is removedfrom the fluidic sample, typically by raising it up and out of thevessel 802 containing the fluidic sample (FIG. 8( c)). In this manner,vessels 802 with differing amounts of fluidic volumes can be accuratelyinterrogated without any previous knowledge of the volume of sample ineach vessel 802. Furthermore, multiple analyses be required from asingle vessel 802, the movement of the sample aspiration tube willautomatically compensate for the reduced amount of sample after eachanalysis.

In another embodiment of the invention, a “safe” level will be set suchthat the sample aspiration tube 407 may not travel below a predetermineddistance. The “safe” level may be determined at manufacture or may beconfigurable to reflect the dimension of particular sample reservoirs.This embodiment protects that sample aspiration tube 407 from makingcontact with the bottom of an empty vessel 802, and in cases in whichthere is no fluidic sample in a given reaction vessel prevents thesample aspiration tube 407 from moving continuously until the bottom ofthe vessel 802 is reached and physical damage to the sample aspirationtube 407 and/or the reaction vessel 802 may occur.

In a further embodiment of the invention, if the sample aspiration tube407 is lowered to the “safe” level without the sensor 702 beingtriggered, the computerized control system will produce an error messageindicating that an aspiration of fluidic sample did not occur. Thiscould be due to several reasons, including but not limited to a vessel802 not containing any fluidic sample, a clog or plug in the sampleaspiration tube 407, or a loss of vacuum. An error message generatedwill permit the user of the invention to pause the analysis and solvethe problem before continuing.

Suitable fluid sensors 702 include optical sensors such as thoseavailable from OPTEK Technology of Carrollton, Tex.

In another embodiment, an optical fluid sensor can be fabricated from alight source, two lengths of fiber optics, and an optical detector. Oneend of the first fiber optic length is coupled with the light source(e.g. a lamp, a red diode laser, and the like). The other end of thefirst fiber optic length is coupled with a first optical window on theconduit, for example, with optical glue. One end of the second fiberoptic length is coupled with a second optical window on the conduit. Theother end of the second fiber optic length is coupled with an opticaldetector, for example, a visible light detector.

FIG. 9 is a flowchart depicting the use of the system described herein.In step S902, the vacuum source 211, 411 is initiated. In step S904,sipper aspiration tube 407 is introduced in sample reservoir 401, 802.In step S906, sensor 702 detects a sample in conduit 704. In step S908,valve 405 is actuated to release the sample in injection loop 403. Instep S910, sipper aspiration tube S910 is retracted from samplereservoir 401, 802. In step S912, wash solution is aspirate throughsample aspiration tube 407 before the process is repeated.

One skilled in the art will appreciate that the steps depicted in FIG. 9need not necessarily performed sequentially. Rather, certain steps maybe performed concurrently, simultaneously, and/or in parallel. Forexample, sample aspiration tube 407 can be retracted from reservoir 401,802 while valve 405 is actuated.

Coupling of Mass Spectrometry Devices with Systems Containing Salts orBuffers

Many biological separations use ion-exchange chromatography (e.g. cationexchange or anion exchange) or size-exclusion chromatography. Thesetechniques have particularly important applications in the separationsof proteins, peptides, oligonucleotides and many other analytes. Theseparation techniques have many applications ranging from scientificresearch and development through the manufacturing of pharmaceuticallyactive compounds.

The techniques typically rely on the selective elution of individualanalytes in a complex mixture from a chromatography matrix in responseto the variation of one or more biophysical parameters. For example, incation exchange chromatography, the concentration of cations in theelution buffer is typically increased in a gradual manner. When theconcentration of cations in the elution buffer reaches a level at whichthe affinity of the cations in the elution buffer for the chromatographymatrix is stronger than the affinity of the analyte, the analyte isdisplaced from the chromatography matrix by the cation. The displacedanalyte is then eluted from the column. Since different analytes withina complex mixture typically have different affinities for thechromatography matrix a separation can be achieved. Other ion exchangesystems rely on a change in pH to enact the desired separation. Manyparameters, such as the selection of chromatography matrix, theselection of the cation used in the elution buffer, the rate at whichcation concentration is varied, and others may need to be optimized inorder to affect a desirable separation.

In size exclusion chromatography (SEC), a separation of analytes isperformed based on the relative size of the analytes. Typical SECseparations are performed using a chromatography matrix that consists ofporous particles. When a mixture of analytes is introduced on thecolumn, smaller analytes tend to travel through the pores in thechromatography matrix, whereas those analytes that are too large areexcluded from the pores and travel through the spaces between theparticles. As a result, large particles tend to have a shorter residencetime in the chromatography matrix and are eluted early. Smallerparticles that travel through the porous matrix have a longer residenceon the column and elute later, thereby enacting a size-based separation.As in ion-exchange chromatography, proper selection of thechromatography matrix, buffers used for eluting the sample, the geometryand size of the separation column, and other factors must be optimizedto achieve a desirable separation outcome.

Both ion-exchange chromatography and SEC require the presence of saltsand/or buffers in the elution fluids. The presence of anions or cationsis particularly unavoidable in the case of ion-exchange chromatographywhere the entire separation is based on the displacement of analytesfrom the chromatography matrix with an ion. However, even in reversedphase chromatography where the elution is generally performed with anorganic solvent and salts are usually not required, there are many caseswhere the separation may be improved through the addition of certainsalts or other compounds to the wash or elution solvents.

Mass spectrometry (MS) is an important analytical technique withapplications including research, drug discovery, environmental testing,forensics, quality control, and many others. Mass spectrometry is amass-selective detector that has the ability to quantitatively detectcompounds based on the molecular mass of the analytes. While manydifferent types of mass spectrometry have been described two main basicapproaches are often used, namely quadrupole and time-of-flight (TOF).Many variations on both of these approaches, including hybrid systemscomprising both approaches, have been developed. In all forms of massspectrometry, the analytes of interest must be ionized and transferredto the gas phase. There are a large number of different methodologiesthat have been employed to achieve this, but most modern systems rely onone of two basic approaches. One approach is atmospheric pressureionization (API), which is further divided into electrospray ionization(ESI) and atmospheric pressure chemical ionization (APCI). The otherapproach is matrix-assisted laser desorption ionization (MALDI).

The various approaches to MS (quadruopole vs. TOF) and sample ionization(API vs. MALDI) have their various strengths and weaknesses for specificapplications. The one constant in all approaches, however, is that MS isnot compatible with analytes that are in solutions that contain highionic strength, such as those that contain high concentrations of saltsor buffers. The presence of high concentrations of ions results in awell-documented phenomenon known as ion suppression. Ion suppressioncauses the analyte of interest to be ionized inefficiently due to theconfounding effect of the high concentration of non-specific ions. Asecond problem with salts and buffers is that many are not volatile. Asa result, the salts tend to deposit on the interior surfaces of the MSsource region and will degrade instrument performance until eventuallythe system is no longer operational.

The incompatibility of MS with samples that contain salts and ionstogether with the need for salts and ions in separation systems such asion exchange and most size-exclusion chromatography applications meansthat the two techniques can not be directly interfaced. There are manycases where it is very advantageous to be able to analyze samplesseparated by ion-exchange or size exclusion chromatography by MS.Currently the only way by which chromatography systems which requiresalts or buffers can be interfaced with MS is to collect the eluate fromthe chromatography in fractions. The fractions are then desalted with asecondary separation process that does not require salts, typically atechnique using a reversed-phase chromatography system. Since a largenumber of fractions may be collected to maintain the fidelity of theseparation process, the secondary desalting process is typically carriedout using a fast system such as solid-phase extraction (SPE) and may beperformed in parallel (e.g. with the use of a 96-well SPE plate). It maybe necessary to concentrate the eluate from the SPE process to increasethe concentration of the analyte(s) to achieve the required sensitivity.The purified, concentrated samples are then analyzed serially with theappropriate MS system. This extends the time and cost associated withthe analysis.

One embodiment of the current invention relates to devices and methodswhich interface a chromatography system that relies on high ionicstrength to achieve separation (such as ion-exchange chromatography)with mass spectrometry. The invention provides a direct and fullyautomated connection between the chromatography system and the MS andeliminates the labor-intensive steps of collecting fractions from thechromatography system, enacting a parallel purification with SPE, sampleconcentration.

The eluate from the chromatography system is connected to an injectionvalve. The injection valve is used to capture an aliquot of the eluatefrom the chromatography system and to divert it to a fast and automatedsample purification system, such as the RAPIDFIRE® system, availablefrom BioTrove, Inc. of Woburn, Mass., which has been described U.S. Pat.No. 6,309,600 to Hunter, U.S. Patent Publication 2002/0001544 of Hess,et al., U.S. Patent Publication 2003/0119193 of Hess, et al., U.S.Patent Publication 2005/0123970 of Ozbal, et al., U.S. Pat. No.6,812,030 to Ozbal, et al., U.S. Pat. No. 6,932,939 to Ozbal, et al.,and U.S. Patent Publication 2005/0194318 of Ozbal, et al. The contentsof the above patents and publications are each incorporated here in itsentirety by reference. The RAPIDFIRE® high throughput mass spectrometrysystem is capable of solid-phase extraction based purification atthroughputs on the order of 5 seconds per sample. With such a system itis possible to take a mass spectrometric reading of the eluate from thechromatography system every 5 seconds. The remaining sample may becollected in fractions for additional analysis or further fractionation.

The general layout of the system is shown in FIG. 10. An injectionsystem 1002 is directly connected to the chromatography system 1004(e.g. a high-pressure liquid chromatography system). Optionally, thechromatography system 1002 may have an optical detector 1006 immediatelyafter the chromatography column to monitor and quantify the analytes asthey elute from the chromatography column. In one embodiment of theinvention, a diversion valve 1008 is placed after the optical detector1006 that may be used to direct the eluate from the chromatographycolumn away from the downstream instrumentation. The diversion valve1008 may be electronically controlled by the optical detector 1006 suchthat if certain signal criteria are met the valve will be actuated. Oneapplication of the diversion valve 1008 may be to divert chromatographyeluate to waste 1010 if the concentration of analytes is too high inorder to protect the injection system 1002 and/or mass spectrometrysystem 1012 from contamination.

In one embodiment of the invention, starting a separation with thechromatographic system generates a trigger signal (e.g. a TTL(transistor-transistor logic) pulse) that is detected by the injectionsystem, the mass spectrometer, the optical detector, and the fractioncollector and is used to synchronize the start of all of the devices.The electronic communication circuitry between the various components ofthe preferred embodiment of the invention is shown as dashed lines inFIG. 10.

Referring now FIG. 11( a), the invention includes two fluidic injectionvalves 1102, 1104 and two high pressure fluidic pumps 1106, 1108. Thefirst pump 1102 is used to flow an aqueous wash solution while thesecond pump 1104 is used to flow an organic elution solution over a SPEcartridge 1110. The details of the SPE cartridge 1112 and itsapplication in sample purification for high-throughput mass spectrometryhave been described previously. See e.g., Nigel J. K. Simpson, SolidPhase Extraction: Principles, Strategies & Applications (2000); E. M.Thurman & M. S. Mills, Solid-Phase Extraction: Principles &Practices(1998). The eluate from the chromatography system 1004 is connecteddirectly to one port of fluidic injection valve 1102 as shown in FIG.11( a). Another tube connected to a second port of the same valve 1102is used to carry the eluate to waste or to a fraction collector 1014,depending on the application. The aqueous fluid from pump 1106 is flowedover the SPE cartridge 1110 in a first direction to condition andequilibrate the cartridge 1110. In the meantime, the organic solventfrom pump 1108 is flowed directly to the source of an API-MS toestablish a stable spray in ESI or APCI mode. This initial fluidiccircuit is shown in FIG. 11( a).

Referring now to FIG. 11( b), once the chromatographic separation isbegun, valve 1102 is electromechanically actuated to the position shownin FIG. 11( b). In this position the eluate from the chromatographysystem 1002 is diverted over an injection loop 1114.

Valve 1102 is actuated a second time to the position shown in FIG. 11(c) after enough time has been allowed to ensure that the injection loop1114 is completely full of sample. The determination of the amount oftime before valve 1102 is actuated the second time can be determined bycalculating the flow rate of the chromatography eluate and the volume ofthe injection loop 1114. For example, if the chromatography solvent ispumped at 0.6 mL/min and a 10 μL injection loop is used, the system willrequire 1 second to completely fill the injection loop (0.6 mL/min=10μL/sec). Alternatively, an optical sensor can be coupled with tube 1112as described above.

Reactuation of valve 1102 after an aliquot of sample has been allowed tofill the loop 1114 as shown in FIG. 11( c) will result in the samplebeing pushed from the loop 1114 onto the SPE cartridge 1110. Theanalytes of interest (e.g. proteins or oliginucleotides) will adsorb onthe SPE cartridge 1110 while the salts and other ions used in thechromatographic separation will pass through the cartridge 1110 and willbe collected in a waste container 1116. Waste container 1116 and 1014can, in various embodiments, be the same or separate vessels.

After approximately 10 SPE column volumes of wash solution have beenflowed over the SPE cartridge 1110, valve 1104 is actuated to theposition shown in FIG. 11( d). In this position, the organic solventfrom the second pump 1108 is diverted over the SPE cartridge 1110 in theopposite direction to the sample loading and washing. The purified anddesalted analyte(s) of interest are solubilized by the organic solvent,desorbed from the SPE cartridge 1110, and flowed onto the API-MS 1118for mass spectrometric analysis. Typically, 10 SPE column volumes oforganic solvent are sufficient to achieve a near complete elution ofanalytes. In a preferred embodiment of the invention, the timing of thewash and elution steps adjustable and may be optimized for each specificapplication. In typical applications, a SPE cartridge with a 4.0 μLcolumn bed volume is used. At a flow rate of 1.2 mL/min (or 20 μL/sec)10 column volumes of wash or elution solvent can be delivered to the SPEcartridge 1110 in as little as 2 seconds.

After the analytes have been delivered to the MS 1118, valve 1104 isactuated again to the position shown in FIG. 11( e). This is the initialposition of the fluidic system and facilitates the reconditioning andequilibration of the SPE cartridge 1110. In a preferred embodiment ofthe invention, the cycle shown in FIGS. 11( a) through 11(e) can berepeated at a rate that is selected by the user through a softwareinterface. For example, the user may select a desalting and MS analysiscycle of 10 seconds. Longer cycle times may be selected, however theminimum cycle time will depend on the timing of the valve cycles.Typical peak widths from ion-exchange or size-exclusion chromatographysystems are in the 10-30 second range meaning that at least one MSanalysis will be available for every peak that is eluted from thechromatography system 1002. Following from the previous example of a 10μL injection loop and a HPLC flow rate of 0.6 mL/min, 10 μL/sec will beflowed over valve 1102 in the desalting apparatus. If 10 μL of sample isremoved via the injection loop at a rate of once per every 10 seconds, atotal of 10% of the total eluate from the HPLC column 1002 will bediverted for rapid desalting and MS analysis. The remaining 90% ofeluate may be collected in a fraction collector or disposed of in awaste container depending on the application.

At the conclusion of the HPLC experiment, a second electronic signalwill direct the injection system 1002, the mass spectrometer 1012, theoptical detector 1006, and the fraction collector 1014 to switch to astandby mode. At the end of the chromatographic separation, the userwill have a continuous optical trace (such as a UV chromatogram)obtained from the optical detector 1006, a series of non-continuous MSdata at the cycling frequency selected, and a series of fractionscollected by the fraction collector. There will be an offset between theoptical detector and the MS data based on the internal volume of thetubing between the optical detector and the MS source and the wash timeselected in the injection system 1002 valving. The offset may becalculated or empirically determined, however, once the offset is knownit will be possible to correctly align the MS data with the optical dataand the appropriate fraction.

With this invention, it is possible to directly collect MS data from aseparation system 1004 that contains MS-incompatible buffers withoutneeding to perform labor intensive and time consuming steps of fractioncollection and off-line sample preparation.

FIG. 12 illustrates a method of processing of an eluted sample from aliquid chromatography system in a mass spectrometry device. In step1202, a flow of a non-polar solvent to the mass spectrometry device isinitiated. In step 1204, an eluted sample is received from the liquidchromatography system. The eluted sample is flowed over an SPE column instep 1206. In step 1208, the SPE column is washed with a polar solution.In step 1210, a non-polar solvent is flowed over the SPE column. In step1212, the non-polar solvent and the eluted sample are presented to themass spectrometry device.

The systems and methods described above can also be reversed so suchthat an eluted sample is non-polar, while the wash solvent is polar. Insuch an embodiment, the column can be a HILIC (Hydrophilic InteractionLiquid Chromatography) column.

Example 4

Cation exchange chromatography is used in the quality control step ofthe manufacturing of a pharmacologically active protein. For each lotthat is manufactured, a 60 minute HPLC separation using an establishedstandard operating procedure must be performed. It is known that theprotein of interest elutes off of the HPLC column between 28 and 30minutes. The entire chromatographic run is monitored by the opticaldetector 1006 at a wavelength of 220 nm. The HPLC 1004 is run at 0.6mL/min and a gradient from 0.1 M sodium chloride to 1M sodium chlorideis used to enact the separation.

If any other chromatographic peaks are detected other than the mainprotein itself it is possible that these are contaminants or breakdownproducts. Because this protein is meant to be administered to patients,it is required that a full characterization of all potential contaminantpeaks be completed before the lot can be approved. Traditionally thischaracterization would involve collecting fractions from the HPLC,performing a sample preparation step to remove the MS-incompatiblesalts, and running the MS measurement. Typically, an aliquot of thefraction must also be re-injected in the HPLC separation to ensure thatthe correct fraction was used in the MS characterization.

The present invention eliminates many of the time consuming and laborintensive steps described above. The device 1002 is placed between theoptical detector 1006 and the fraction collector 1014 as shown in FIG.10. A 10 μL injection loop 1114 is used along with a SPE cartridge 1110that contains a polymeric matrix with a 4.0 μL bed volume. Pump 1102 isused to deliver a wash solvent consisting of water with 0.02%trifluoroacetic acid while pump 1104 is used to deliver an elutionsolvent of 80% acetonitrile with 0.02% trifluoroacetic acid. A time of 1second is selected to completely fill the injection loop 1114, 2 secondsto wash the salts away from the analytes, 2 seconds to elute theanalytes off of the SPE cartridge 1110, and 1 second to fullyrecondition the SPE cartridge 1110. For this application it is decidedto run the system at the fastest cycle time, which is 6 seconds.

When the cation exchange HPLC run is initiated, a TTL pulse alsotriggers the start of the UV detector 1006, the MS 1012, the injectionsystem 1002, and the fraction collector 1014. Over the 60 minute, run atotal of 600 high throughput mass spectrometry system cycles will beperformed (3600 seconds at 6 seconds/cycle=600 cycles). At the end ofthe experiment, the MS data, consisting of a time trace with 600injections can be aligned with the optical detector either through theidentification of a landmark (e.g. the main protein in the assay) orthrough the calculation of the delay within the system. Using theinvention described, the equivalent experiment to collecting andpreparing 600 individual fractions from the HPLC can be performed in acompletely automated fashion and obviate the need for any additionalvalidation experiments.

The embodiments of the invention described herein can be controlled by avariety of electronic devices including hardware and software as isknown to those of skill in the art. Electrical-mechanical componentssuch as valves 206, 207, 601, 602, 603, 1102, 1104 can be controlledaccording to interfaces described in related literature can communicatewith control devices according to a variety of standard and proprietarytechnologies and protocols including, but not limited to,transistor-transistor logic (TTL), serial, parallel, FireWire, USB,Ethernet, and the like.

The functions of several elements may, in alternative embodiments, becarried out by fewer elements, or a single element. Similarly, in someembodiments, any functional element may perform fewer, or different,operations than those described with respect to the illustratedembodiment. Also, functional elements (e.g., modules, databases,computers, clients, servers and the like) depicted as distinct forpurposes of illustration may be incorporated within other functionalelements, separated in different hardware or distributed in a particularimplementation.

While certain embodiments according to the invention have beendescribed, the invention is not limited to just the describedembodiments. Various changes and/or modifications can be made to any ofthe described embodiments without departing from the spirit or scope ofthe invention. Also, various combinations of elements, steps, features,and/or aspects of the described embodiments are possible andcontemplated even if such combinations are not expressly identifiedherein.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications, andother references cited herein are hereby expressly incorporated hereinin their entireties by reference.

1. A method of preparing an eluted sample containing salts or buffersfrom a liquid chromatography device for analysis by a mass spectrometrydevice, the method comprising: continuously providing a non-polarsolvent to the mass spectrometry device; receiving the eluted samplefrom the liquid chromatography device; flowing the eluted sample over asolid phase extraction column; flowing the non-polar solvent over thesolid phase extraction column; and presenting the non-polar solvent andthe eluted sample to the mass spectrometry device.
 2. The method ofclaim 1, wherein the liquid chromatography device is an ion exchangechromatography device.
 3. The method of claim 1, wherein the liquidchromatography device is a cation exchange chromatography device.
 4. Themethod of claim 1, wherein the liquid chromatography device is a sizeexclusion chromatography device.
 5. The method of claim 1, furthercomprising: optically analyzing the eluted sample from the liquidchromatography device to generate an optical data set.
 6. The method ofclaim 5, further comprising: associating the optical data set with adata set generated by the mass spectrometry device.
 7. The method ofclaim 1 further comprising: flowing a polar wash solution over the solidphase extraction column.
 8. A sample injection system for coupling aliquid chromatography device with a mass spectrometry device, the systemcomprising: a sample injection valve having: i. a first position thatallows sample from the liquid chromatography device to pass through thesample injection system, and ii. a second position that loads samplefrom the liquid chromatography device onto a sample supply loop; and acolumn control valve configured to facilitate a continuous flow of anelution solvent to a sample analyzer, the column control valve having:i. a first position that simultaneously delivers the fluidic sample fromthe sample supply loop to a solid phase extraction column in a firstdirection and delivers an elution solvent to the sample analyzer, andii. a second position that flows the elution solvent over the solidphase extraction column in a second direction to deliver the fluidicsample and the elution solvent to the sample analyzer.
 9. The system ofclaim 8, further comprising: an optical detector for analyzing thesample from the liquid chromatography device.
 10. The system of claim 8,further comprising: a diversion valve located between the liquidchromatography device and the sample injection valve.
 11. The system ofclaim 10, wherein the diversion valve is actuated as a result of signalgenerated by the optical detector.
 12. The system of claim 8, furthercomprising a fraction collector.
 13. The system of claim 8, wherein theelution solvent is a polar solvent.
 14. The system of claim 8, whereinthe elution solvent is a non-polar solvent.
 15. A method of preparing aneluted sample from a liquid chromatography device for analysis by a massspectrometry device, the method comprising: continuously providing apolar solvent to the mass spectrometry device; receiving the elutedsample from the liquid chromatography device; flowing the eluted sampleover a HILIC column; flowing the polar solvent over the HILIC column;and presenting the polar solvent and the eluted sample to the massspectrometry device.
 16. The method of claim 15, wherein the liquidchromatography device is one selected from the group consisting of: anion exchange chromatography device, a cation exchange chromatographydevice, and a size exclusion chromatography device.
 17. The method ofclaim 13, further comprising: optically analyzing the eluted sample fromthe liquid chromatography device to generate an optical data set. 18.The method of claim 17, further comprising: associating the optical dataset with a data set generated by the mass spectrometry device.
 19. Themethod of claim 1 further comprising: flowing a polar wash solution overthe solid phase extraction column.